Compositions and methods relating to universal glycoforms for enhanced antibody efficacy

ABSTRACT

The present disclosure relates to compositions and methods of use comprising antibodies or binding fragments thereof further comprising universal Fc glycoforms.

RELATED APPLICATION

This application claims the benefit of priority of, and is aContinuation-in-Part of, U.S. application Ser. No. 14/723,297, filed onMay 27, 2015. The instant application also claims priority to, and is aContinuation-in-Part of, U.S. application Ser. No. 14/798,312, filed onJul. 13, 2015. This application also claims priority to U.S. applicationSer. No. 62/110,338, filed on Jan. 30, 2015; U.S. application Ser. No.62/003,136, filed on May 27, 2014; U.S. application Ser. No. 62/003,104,filed on May 27, 2014; U.S. application Ser. No. 62/003,908, filed onMay 28, 2014; and U.S. application Ser. No. 62/020,199, filed on Jul. 2,2014. The content of each of which is incorporated herein.

FIELD

The present disclosure relates to selected universal Fc glycoforms tunedto the desired binding/effector activity for enhancing the therapeuticefficacies of antibodies directed against many diseases, includingcancers, inflammatory disorders and infectious diseases. Particularly,the selected and/or directed optimized universal Fc glycoforms can begenerated and/or incorporated to the design and/or the generation ofmonoclonal antibodies for enhanced therapeutic efficacy.

BACKGROUND

Antibody-based therapies have a proven record of efficacy against manydiseases including inflammatory disorders, cancers, infectious diseases,and solid organ transplant rejection. Currently, more than 40therapeutic monoclonal antibodies (mAbs) are approved for clinical usein USA, EU and several other countries. Most of those are for therapy ofcancer and immune diseases. Examples of therapeutic antibodies withanti-tumor activities include anti-CD20, anti-Her2, anti-EGFR,anti-CD40, anti-CTLA-4, and anti-PD-1 antibodies.

The majority of approved biopharmaceuticals are produced in mammaliancell culture systems to deliver proteins with desired glycosylationpatterns and thus ensure reduced immunogenicity and higher in vivoefficacy and stability. Non-human mammalian expression systems such asCHO or NS0 cells have the machinery required to add complex, human-typeglycans. However, glycans produced in these systems can differ fromglycans produced in humans. Their glycosylation machinery often addsundesired carbohydrate determinants which may alter protein folding,induce immunogenicity, and reduce circulatory life span of the drug.

Furthermore, mammalian cell culture delivers a heterogeneous mixture ofglycosylation patterns which do not all have the same properties.Properties like safety, efficacy and the serum half-life of therapeuticproteins can be affected by these glycosylation patterns. The mammaliancell culture system delivers heterogeneous mixtures of glycosylationpatterns which do not all have the same properties.

SUMMARY

Fc glycosylation has been an important subject in the field oftherapeutic monoclonal antibodies. Fc glycosylation can significantlymodify Fc effector functions such as Fc receptor binding and complementactivation, and thus affect the in vivo safety and efficacy profiles oftherapeutic antibodies. Diversity in Fc glycosylation within an antibodywill correspond to diversity in Fc effector functions. Thus, thisheterogeneity in Fc glycans has a functional consequence as itinfluences binding of IgG molecules to Fc receptors and thereby impactsantibody effector functions, and may trigger undesired effects inpatients thus deeming them a safety concern.

There is a need for improved monoclonal antibody therapy against manydiseases including inflammatory disorders, cancers and infectiousdiseases. Some specific glycoforms in Fc can confer desired biologicalfunctions with improved effector functions, such as antibody-dependentcellular cytotoxicity (ADCC). Thus, it is useful to generate therapeuticantibodies with optimized Fc glycoforms.

Accordingly, the present disclosure provide selected universal Fcglycoforms tuned to the desired binding/effector activity for enhancingthe efficacy of therapeutic antibodies against many diseases, includingcancers, inflammatory disorders and infectious diseases. The selectedand/or directed optimized universal Fc glycoforms can be applied and/orincorporated to the design and/or the generation of monoclonalantibodies (preferably, therapeutic monoclonal antibodies) for enhancedtherapeutic efficacy.

In one aspect, the present disclosure provided a Fc glycoform forenhancing binding/effector activity in monoclonal antibody, wherein saidantibody comprising a glycoform having the formula:

Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ (see FIG. 1)

In some embodiments, the present disclosure provided a pharmaceuticalcomposition comprising the glycoform of FIG. 1 and a pharmaceuticallyacceptable carrier. In one aspect, the present disclosure provided amethod of treating an infectious, hyperproliferative disease and/orcondition, wherein the method comprises administering to a subject inneed thereof a pharmaceutical composition comprising the glycoformhaving the Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2

In some embodiments, the antibody is a mouse, chimeric, humanized,and/or human MC41 antibody comprising the following sequences:

TABLE 1-1 Amino acid and nucleotide sequencesof anti-SSEA-4 murine, MC41. SEQ ID NO DESCRIPTION SEQUENCE 200 MC41 VHCAGGTGCAGCTGAAGGAAAGCGGACCCG nucleotide GACTGGTCGCCCCCTCTAAGTCTCTGTCsequence TATTACTTGTACTGTGAGCGGATTCTCT CTGAGCTCCCAGGGCGTGTACTGGGTGAGGCAGCCACCTGGCAAGGGCCTGGAGTG GCTGGGAGCCATCTGGGCAGGAGGCAGCACCAACTATAATTCCGCCCTGATGTCTC GCCTGTCTATCAGCAAGGACAACTCCAAGTCTCAGGTGTTCCTGAAGATGAACAGC CTGCAGACCGACGATACAGCCATGTACTATTGCGCCCGGGTGGACGGCTACAGAGG CTATAACATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC 201 MC41 VL GAGAATGTGCTGACACAGTCCCCAGCAAnucleotide TCATGAGCGCCTCCCCAGGAGAGAAGGT sequenceGACCATGACATGTTCCGCCTCCTCTAGC GTGTCTTACATGCACTGGTATCAGCAGAAGTCCTCTACCAGCCCTAAGCTGTGGAT CTACGACACAAGCAAGCTGGCCTCCGGCGTGCCCGGCCGGTTTTCTGGCAGCGGCT CCGGCAACTCTTATAGCCTGACCATCAGCAGCATGGAGGCCGAGGATGTGGCCACA TACTATTGCTTTCAGGGCTCTGGCTACCCACTGACATTCGGGGCTGGAACTAAACT GGAACTGAAGCGA 202 MC41 VHQVQLKESGPGLVAPSKSLSITCTVSGFS amino acid LSSQGVYWVRQPPGKGLEWLGAIWAGGSsequence TNYNSALMSRLSISKDNSKSQVFLKMNS LQTDDTAMYYCARVDGYRGYNMDYWGQGTSVTVSS 203 MC41 VL ENVLTQSPAIMSASPGEKVTMTCSASSS amino acidVSYMHWYQQKSSTSPKLWIYDTSKLASG sequence VPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGAGTKLELKR 204 MC41 VL SSVSY CDR1 205 MC41 VL DTS CDR2 206MC41 VL FQGSGYPLT CDR3 207 MC41 VH GFSLSSQG CDR1 208 MC41 VH IWAGGSTCDR2 209 MC41 VH ARVDGYRGYNMDY CDR3

TABLE 1-2 Amino acid and nucleotide sequences of 2^(nd)humanized monoclonal antibody, hMC41. 2^(nd) SEQ ID NO DESCRIPTIONSEQUENCE 210 MC41 VH CAGGTGCAGCTGAAGGAGTCCGGACCAG nucleotideGACTGGTGGCACCATCTAAGACCCTGAG sequence CCTGACCTGCACAGTGAGCGGCTTCTCCCTGAGCTCCCAGGGCGTGTACTGGATCA GGCAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGCCATCTGGGCCGGCGGCTCT ACAAACTATAATTCCGCCCTGATGTCTCGCCTGTCTATCAGCAAGGACAACTCCAA GTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATGTACT ATTGCGCCCGGGTGGACGGCTACAGAGGCTATAACATGGATTATTGGGGCCAGGGC ACCCTGGTGACAGTGTCTAGC 211 MC41 VLGAGAATGTGCTGACCCAGTCTCCTGCCA nucleotide TCATGAGCGCCACACCAGGCGAGAAGGTsequence GACCATGACATGTTCCGCCTCCTCTAGC GTGTCTTACCTGCACTGGTATCAGCAGAAGTCCTCTACCAGCCCCAAGCTGTGGAT CTACGACACAAGCAAGCTGGCATCCGGAGTGCCTGGCCGGTTCAGCGGATCCGGAT CTGGAAACAGCTATACCCTGACAATCAGCTCCATGGAGGCCGAGGATGTGGCCACC TACTATTGTTTCCAGGGATCCGGATACCCACTGACCTTTGGCGCCGGCACAAAGCT GGAGATCAAGCGT 212 MC41 VHQVQLKESGPGLVAPSKTLSLTCTVSGFS amino acid LSSQGVYWIRQPPGKGLEWIGAIWAGGSsequence TNYNSALMSRLSISKDNSKSQVFLKMNS LQTDDTAMYYCARVDGYRGYNMDYWGQGTLVTVSS 213 MC41 VL ENVLTQSPAIMSATPGEKVTMTCSASSS amino acidVSYLHWYQQKSSTSPKLWIYDTSKLASG sequence VPGRFSGSGSGNSYTLTISSMEAEDVATYYCFQGSGYPLTFGAGTKLEIKR 214 MC41 VL SSVSY CDR1 215 MC41 VL DTS CDR2 216MC41 VL FQGSGYPLT CDR3 217 MC41 VH GFSLSSQG CDR1 218 MC41 VH IWAGGSTCDR2 219 MC41 VH ARVDGYRGYNMDY CDR3

TABLE 1-3 Amino acid and nucleotide sequences of 3^(rd)humanized monoclonal antibody, hMC41. 3^(rd) SEQ ID NO DESCRIPTIONSEQUENCE 220 MC41 VH CAGGTGCAGCTGAAGGAGTCCGGACCAG nucleotideGACTGGTGGCACCATCTAAGACCCTGAG sequence CCTGACCTGCACAGTGAGCGGCTTCTCCCTGAGCTCCCAGGGCGTGTACTGGATCA GGCAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGCCATCTGGGCCGGCGGCTCT ACAAACTATAATTCCGCCCTGATGTCTCGCCTGTCTATCAGCAAGGACAACTCCAA GTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGACGATACAGCCATGTACT ATTGCGCCCGGGTGGACGGCTACAGAGGCTATAACATGGATTATTGGGGCCAGGGC ACCtcGGTGACAGTGTCTAGC 221 MC41 VLGAGAATGTGCTGACCCAGTCTCCTGCCA nucleotide TCATGAGCGCCACACCAGGCGAGAAGGTsequence GACCATGACATGTTCCGCCTCCTCTAGC GTGTCTTACATGCACTGGTATCAGCAGAAGTCCTCTACCAGCCCCAAGCTGTGGAT CTACGACACAAGCAAGCTGGCATCCGGAGTGCCTGGCCGGTTCAGCGGATCCGGAT CTGGAAACAGCTATACCCTGACAATCAGCTCCATGGAGGCCGAGGATGTGGCCACC TACTATTGTTTCCAGGGATCCGGATACCCACTGACCTTTGGCGCCGGCACAAAGCT GGAGATCAAGCGT 222 MC41 VHQVQLKESGPGLVAPSKTLSLTCTVSGFS amino acid LSSQGVYWIRQPPGKGLEWIGAIWAGGSsequence TNYNSALMSRLSISKDNSKSQVFLKMNS LQTDDTAMYYCARVDGYRGYNMDYWGQGTSVTVSS 223 MC41 VL ENVLTQSPAIMSATPGEKVTMTCSASSS amino acidVSYMHWYQQKSSTSPKLWIYDTSKLASG sequence VPGRFSGSGSGNSYTLTISSMEAEDVATYYCFQGSGYPLTFGAGTKLEIKR 224 MC41 VL SSVSY CDR1 225 MC41 VL DTS CDR2 226MC41 VL FQGSGYPLT CDR3 227 MC41 VH GFSLSSQG CDR1 228 MC41 VH IWAGGSTCDR2 229 MC41 VH ARVDGYRGYNMDY CDR3

In one aspect, the present disclosure provides an isolated monoclonalantibody or a binding fragment thereof that binds toNeu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 wherein the antibody orthe fragment thereof comprises a Fc glycoform for enhancingbinding/effector activity in monoclonal antibody, wherein said antibodycomprising a glycoform having the formula:

Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ (see FIG. 1)

In one embodiment, the antibody is an IgG1 and the binding toNeu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→ is specific binding.

In one embodiment, the antibody comprised VH having SEQ ID NO: 147 orSEQ ID No:137 and VL having SEQ ID No: 148 or SEQ ID No:138.

In one embodiment, the isolated antibody, or antigen-binding fragmentthereof comprising H-CDR1, H-CDR2, and H-CDR3 selected from (i)-(iii):

-   -   (i) H-CDR1 selected from SEQ ID NO:152 (GFSLTSYG);    -   (ii) H-CDR2 selected from SEQ ID NO: 153 (IWGEGST);    -   (iii) H-CDR3 selected from SEQ ID NO:154 (AMTGTAY),        respectively;    -   and comprising L-CDR1, L-CDR2 and L-CDR3 selected from        (iv)-(vi):    -   (iv) L-CDR1 selected from SEQ ID NO: 149 (SSVSY);    -   (v) L-CDR2 selected from SEQ ID NO:150 (DTS); and    -   (vi) L-CDR3 selected from SEQ ID NO: 151(HQWSSSPHT),        respectively.

In one embodiment, the isolated antibody or antigen-binding fragmentfurther comprising H-FR1, H-FR2, H-FR3, and HFR4 selected from (i)-(iv):

-   -   (i) H-FR1 selected from SEQ ID NO:159        (QVQLKESGPGLVAPSQSLSITCTVS);    -   (ii) H-FR2 selected from SEQ ID NO:160 (VSWIRQPPGKGLEWIGV);    -   (iii) H-FR3 selected from SEQ ID NO:161        (NYHSVLISRLTISKDNSKSQVFLKLNSLQTDDTATYYC);    -   (iv) H-FR4 selected from SEQ ID NO:162 (WGQGTLVTVSS);        respectively;    -   and comprising L-FR1, L-FR2, L-FR3 and L-FR4 selected from        (v)-(viii):

(v) L-FR1 selected from SEQ ID NO: 155 (QIVLTQSPAIMSASPGEKVTMTCSAS);

(vi) L-FR2 selected from SEQ ID NO:156 (MHWYQQKSGTSPKRWIY);

(vii) L-FR3 selected from SEQ ID NO: 157(KLSSGVPGRFSGSGSGTSYSLTISRLEAEDAATYYC);

(viii) L-FR4 selected from SEQ ID NO: 158 (FGGGTKVEIKR); respectively.

In one embodiment, the antibody is a human antibody.

In one embodiment, the antibody is a humanized antibody.

In one embodiment, the antibody comprised VH having SEQ ID NO: 200, SEQID No. 210 or SEQ ID No:137 and VL having SEQ ID No: 201 SEQ ID No. 211or SEQ ID No: 221.

In one embodiment, the isolated antibody, or antigen-binding fragmentthereof comprises H-CDR1, H-CDR2, and H-CDR3 selected from (i)-(iii):

-   -   (i) H-CDR1 selected from SEQ ID NO:207, SEQ ID NO: 217, SEQ ID        NO: 227;    -   (ii) H-CDR2 selected from SEQ ID NO: 208; SEQ ID NO: 218, SEQ ID        NO: 228;    -   (iii) H-CDR3 selected from SEQ ID NO: 209, SEQ ID NO: 219, SEQ        ID NO: 229; respectively;    -   and comprising L-CDR1, L-CDR2 and L-CDR3 selected from        (iv)-(vi):    -   (iv) L-CDR1 selected from SEQ ID NO: SEQ ID NO: 204; SEQ ID NO:        214, and SEQ ID NO: 224;    -   (v) L-CDR2 selected from SEQ ID NO:205; SEQ ID NO: 215 and SEQ        ID NO: 225;    -   (vi) L-CDR3 selected from SEQ ID NO: 206, SEQ ID NO: 216 and SEQ        ID NO: 226;    -   respectively.

In one embodiment, the antibody of claim 9 wherein the antibody is ahuman antibody.

In one embodiment, the antibody of claim 9 wherein the antibody is ahumanized antibody.

In one embodiment, the antigen binding fragment is a Fab fragment, aF(ab′)2 fragment, or a single-chain Fv fragment.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising the monoclonal antibody or binding fragmentthereof of any one of claim 6, 7, 10, or 11 and a pharmaceuticallyacceptable carrier.

In one embodiment, the pharmaceutical composition is useful in thetreatment against a hyperproliferative disease.

In one asepct, the present disclosure provides a method of treatingcancer in a subject in need thereof, wherein the method comprisesadministering to the subject a therapeutically effective amount of thepharmaceutical composition of claim 13 whereby the administered antibodyenhances ADCC activity in said subject.

In one embodiment, the method of treatment for cancer is selected fromthe group consisting of brain cancer, lung cancer, breast cancer, oralcancer, esophageal cancer, stomach cancer, liver cancer, bile ductcancer, pancreatic cancer, colon cancer, kidney cancer, bone cancer,skin cancer, cervical cancer, ovarian cancer, and prostate cancer.

In one embodiment, the method comprising optionally administering acombined pharmaceutical formulation with at least one otherchemotherapeutic agent.

In another aspect, the present disclosure also provides a method formaking a population of homogeneous antibodies comprising:

(a) contacting a monoclonal antibody with an α-fucosidase and at leastone endoglycosidase;

(b) generating a defucosylated antibody having a singleN-acetylglucosamine (GlcNAc); and (c) adding the universal glycan toGlcNAc of Fc region of antibody to form the homogeneous antibody withsaid glycoform.

In one embodiment, the antibody or binding fragment thereof includesantibodies or binding fragments thereof specifically bind to one or moreof the antigens selected from the group consisting of Globo H, SSEA-3and SSEA-4.

One other aspect of the present disclosure provides humanizedglycoantibodies based on the modification of the MC48. Exemplars andtheir amino acid and nucleic acid structures/sequences are providedbelow:

TABLE 17-0 Amino Acid and Nucleotide Sequencesof Mouse Monoclonal Antibody MC48. SEQ ID NO DESCRIPTION SEQUENCE 41MC48 VH CAGGTGCAGCTGAAGGAGTCAGGACCTG nucleotideGCCTGGTGGCGCCCTCACAGAGCCTGTC sequence CATCACATGCACTGTCTCAGGGTTCTCATTAACCAGCTATGGTGTAAGCTGGGTTC GCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTGAGGGGAGC ACAAATTATCATTCAGTTCTCATATCCAGACTGACCATTAGTAAGGATAACTCCAA GAGCCAAGTTTTCTTAAAACTGAACAGTCTGCAAACTGATGACACAGCCACGTACT ACTGTGCCATGACTGGGACAGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT GCA 42 MC48 VL CAAATTGTTCTCACCCAGTCTCCAGCAAnucleotide TCATGTCTGCATCTCCAGGGGAGAAGGT sequenceCACCATGACCTGCAGTGCCAGCTCAAGT GTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGAT TTATGACACATCCAAACTGTCTTCTGGAGTCCCTGGTCGCTTCAGTGGCAGTGGGT CTGGGACCTCTTACTCTCTCACAATCAGCAGGTTGGAGGCTGAAGATGCTGCCACT TATTACTGCCATCAGTGGAGTAGTAGTCCACACACGTTCGGAGGGGGGACCAAGTT GGAGATAAAA 43 MC48 VHQVQLKESGPGLVAPSQSLSITCTVSGFS amino acid LTSYGVSWVRQPPGKGLEWLGVIWGEGSsequence TNYHSVLISRLTISKDNSKSQVFLKLNS LQTDDTATYYCAMTGTAYWGQGTLVTVS A 44MC48 VL QIVLTQSPAIMSASPGEKVTMTCSASSS amino acidVSYMHWYQQKSGTSPKRWIYDTSKLSSG sequence VPGRFSGSGSGTSYSLTISRLEAEDAATYYCHQWSSSPHTFGGGTKLEIK 45 MC48 VL SSVSY CDR1 46 MC48 VL DTS CDR2 47MC48 VL HQWSSSPHT CDR3 48 MC48 VH GFSLTSYG CDR1 49 MC48 VH IWGEGST CDR250 MC48 VH AMTGTAY CDR3

TABLE 17-1 Amino Acid and Nucleotide Sequencesof Humanized Monoclonal Antibody MC48 (1^(st)) SEQ ID NO DESCRIPTIONSEQUENCE 115 hMC48 VH CAGGTGCAGCTGCAAGAGTCAGGACCTG nucleotideGCCTGGTGAAACCCTCAGAAACTCTGTC sequence CCTTACATGCACTGTCTCAGGGTTCTCATTAACCAGCTATGGTGTAAGCTGGATTC GCCAGCCTCCAGGAAAGGGTCTGGAGTGGATTGGAGTAATATGGGGTGAGGGGAGC ACAAATTATCATTCAGTTCTCATATCCAGACTGACCATTAGTGTGGATACCTCCAA GAATCAATTTAGCTTAAAACTGAGCAGTGTTACCGCTGCTGACACAGCCGTTTACT ACTGTGCCATGACTGGGACAGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT AGC 116 hMC48 VLGAGATTGTGCTGACCCAGAGCCCTGCCA nucleotide CACTGTCACTGAGCCCAGGCGAGCGAGCsequence CACACTGTCCTGTTCTGCTAGCTCCTCT GTCTCCTACATGCATTGGTATCAGCAGAAGCCAGGACTGGCACCACGACTGCTGAT CTATGACACTTCTAAACTGAGTTCAGGCATTCCCGCCAGATTCAGTGGCTCAGGGA GCGGAACCGACTTTACTCTGACCATTAGCTCCCTGGAGCCTGAAGATTTCGCCGTG TACTATTGCCATCAGTGGTCATCAAGCCCTCATACCTTCGGGGGGGGGACTAAGGT GGAAATCAAACGC 117 hMC48 VHQVQLQESGPGLVKPSETLSLTCTVSGFS amino acid LTSYGVSWIRQPPGKGLEWIGVIWGEGSsequence TNYHSVLISRLTISVDTSKNQFSLKLSS VTAADTAVYYCAMTGTAYWGQGTLVTVS S 118hMC48 VL EIVLTQSPATLSLSPGERATLSCSASSS amino acidVSYMHWYQQKPGLAPRLLIYDTSKLSSG sequence IPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQWSSSPHTFGGGTKVEIKR 119 hMC48 VL SSVSY CDR1 120 hMC48 VL DTS CDR2121 hMC48 VL HQWSSSPHT CDR3 122 hMC48 VH GFSLTSYG CDR1 123 hMC48 VHIWGEGST CDR2 124 hMC48 VH AMTGTAY CDR3

TABLE 17-2 Amino Acid and Nucleotide Sequencesof Humanized Monoclonal Antibody MC48 (2^(nd)) SEQ ID NO DESCRIPTIONSEQUENCE 125 hMC48 VH CAGGTGCAGCTGAAGCAGAGCGGACCTG nucleotideGCCTGGTGCAGCCCTCACAGAGCCTGAG sequence CATCACTTGTACCGTCAGTGGATTCTCCCTGACATCTTACGGCGTGTCTTGGGTCA GGCAGAGCCCTGGCAAGGGGCTGGAGTGGCTGGGCGTGATCTGGGGAGAAGGCTCA ACTAACTATCACAGCGTCCTGATCAGTCGCCTGTCAATTAACAAGGACAATTCTAA AAGTCAGGTGTTCTTTAAAATGAACAGCCTGCAGTCCAATGATACCGCCATCTACT ATTGCGCTATGACCGGCACAGCATACTGGGGGCAGGGAACACTGGTGACTGTCTCC GCT 126 hMC48 VLGAGATTGTGCTGACCCAGAGCCCTGCCA nucleotide CACTGTCACTGAGCCCAGGCGAGCGAGCsequence CACACTGTCCTGTTCTGCTAGCTCCTCT GTCTCCTACATGCATTGGTATCAGCAGAAGCCAGGACTGGCACCACGACTGCTGAT CTATGACACTTCTAAACTGAGTTCAGGCATTCCCGCCAGATTCAGTGGCTCAGGGA GCGGAACCGACTTTACTCTGACCATTAGCTCCCTGGAGCCTGAAGATTTCGCCGTG TACTATTGCCATCAGTGGTCATCAAGCCCTCATACCTTCGGGGGGGGGACTAAGCT GGAAATCAAACGC 127 hMC48 VHQVQLKQSGPGLVQPSQSLSITCTVSGFS amino acid LTSYGVSWVRQSPGKGLEWLGVIWGEGSsequence TNYHSVLISRLSINKDNSKSQVFFKMNS LQSNDTAIYYCAMTGTAYWGQGTLVTVS A 128hMC48 VL EIVLTQSPATLSLSPGERATLSCSASSS amino acidVSYMHWYQQKPGLAPRLLIYDTSKLSSG sequence IPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQWSSSPHTFGGGTKVLEIKR 129 hMC48 VL SSVSY CDR1 130 hMC48 VL DTS CDR2131 hMC48 VL HQWSSSPHT CDR3 132 hMC48 VH GFSLTSYG CDR1 133 hMC48 VHIWGEGST CDR2 134 hMC48 VH AMTGTAY CDR3

TABLE 17-3 Amino Acid and Nucleotide Sequencesof Humanized Monoclonal Antibody MC48 (3^(rd)) SEQ ID NO DESCRIPTIONSEQUENCE 135 hMC48 VH CAGGTGCAGCTGCAGGAAAGCGGACCCG nucleotideGACTGGTGAAACCTAGCGAAACACTGAG sequence CCTGACTTGTACCGTGAGCGGATTTTCCCTGACCTCTTATGGAGTGAGCTGGATCA GACAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCGTGATTTGGGGAGAAGGCTCC ACAAACTATCACAGTGTCCTGATCTCACGACTGACTATTTCTAAGGACAACTCTAA AAGTCAGGTCTTCCTGAAACTGAATAGTCTGCAGACTGACGATACCGCTACATACT ATTGCGCAATGACAGGGACAGCATACTGGGGACAGGGAACCCTGGTGACAGTCAGC TCC 136 hMC48 VLCAGATCGTGCTGACACAGTCCCCTGCAA nucleotide TTATGTCAGCCAGCCCAGGGGAAAAGGTsequence GACAATGACTTGTAGTGCTTCTAGTTCA GTCTCATACATGCATTGGTATCAGCAGAAGCCAGGCCTGGCCCCCAGACTGCTGAT CTACGACACCTCCAAACTGAGCTCCGGCGTGCCCGGGAGATTTTCCGGCTCTGGGA GTGGAACTTCATATAGCCTGACCATTTCTAGGCTGGAGGCCGAAGATGCCGCTACA TACTATTGCCACCAGTGGAGCAGTAGCCCCCATACATTCGGAGGCGGGACCAAAGT GGAAATCAAACGC 137 hMC48 VHQVQLQESGPGLVKPSETLSLTCTVSGFS amino acid LTSYGVSWIRQPPGKGLEWIGVIWGEGSsequence TNYHSVLISRLTISKDNSKSQVFLKLNS LQTDDTATYYCAMTGTAYWGQGTLVTVS S 138hMC48 VL QIVLTQSPAIMSASPGEKVTMTCSASSS amino acidVSYMHWYQQKPGLAPRLLIYDTSKLSSG sequence VPGRFSGSGSGTSYSLTISRLEAEDAATYYCHQWSSSPHTFGGGTKVEIKR 139 hMC48 VL SSVSY CDR1 140 hMC48 VL DTS CDR2141 hMC48 VL HQWSSSPHT CDR3 142 hMC48 VH GFSLTSYG CDR1 143 hMC48 VHIWGEGST CDR2 144 hMC48 VH AMTGTAY CDR3

TABLE 17-4 Amino Acid and Nucleotide Sequencesof Humanized Monoclonal Antibody MC48 (4^(th)) SEQ ID NO DESCRIPTIONSEQUENCE 145 hMC48 VH CAGGTCCAGCTGAAAGAGAGCGGCCCCG nucleotideGACTGGTCGCCCCTTCACAGAGCCTGAG sequence CATTACTTGCACCGTGAGCGGATTTTCACTGACCAGCTACGGAGTGAGCTGGATTA GACAGCCTCCTGGCAAGGGACTGGAGTGGATCGGCGTGATTTGGGGAGAAGGCAGC ACCAACTATCACAGTGTCCTGATCTCACGCCTGACAATTTCCAAGGACAACAGCAA ATCCCAGGTCTTCCTGAAACTGAATTCTCTGCAGACTGACGATACCGCTACATACT ATTGCGCAATGACAGGGACAGCATACTGGGGACAGGGAACCCTGGTGACAGTCAGT AGT 146 hMC48 VLCAGATCGTGCTGACACAGTCCCCAGCAA nucleotide TTATGTCTGCCAGTCCCGGGGAGAAGGTsequence GACAATGACTTGTAGTGCCAGCTCCTCT GTCTCATACATGCATTGGTATCAGCAGAAGTCCGGCACATCTCCTAAACGGTGGAT CTACGACACTTCTAAACTGAGTTCAGGCGTGCCCGGGAGATTTTCAGGCAGCGGGT CCGGAACTTCTTATAGTCTGACCATTTCCCGACTGGAGGCCGAAGATGCCGCTACC TACTATTGCCATCAGTGGTCTTCAAGCCCTCATACTTTTGGGGGGGGAACTAAGGT GGAAATCAAGCGA 147 hMC48 VHQVQLKESGPGLVAPSQSLSITCTVSGFS amino acid LTSYGVSWIRQPPGKGLEWIGVIWGEGSsequence TNYHSVLISRLTISKDNSKSQVFLKLNS LQTDDTATYYCAMTGTAYWGQGTLVTVS S 148hMC48 VL QIVLTQSPAIMSASPGEKVTMTCSASSS amino acidVSYMHWYQQKSGTSPKRWIYDTSKLSSG sequence VPGRFSGSGSGTSYSLTISRLEAEDAATYYCHQWSSSPHTFGGGTKVEIKR 149 hMC48 VL SSVSY CDR1 150 hMC48 VL DTS CDR2151 hMC48 VL HQWSSSPHT CDR3 152 hMC48 VH GFSLTSYG CDR1 153 hMC48 VHIWGEGST CDR2 154 hMC48 VH AMTGTAY CDR3 155 hMC48 VLQIVLTQSPAIMSASPGEKVTMTCSAS FR1 156 hMC48 VL MHWYQQKSGTSPKRWIY FR2 157hMC48 VL KLSSGVPGRFSGSGSGTSYSLTISRLEA FR3 EDAATYYC 158 hMC48 VLFGGGTKVEIKR FR4 159 hMC48 VH QVQLKESGPGLVAPSQSLSITCTVS FR1 160 hMC48 VHVSWIRQPPGKGLEWIGV FR2 161 hMC48 VH NYHSVLISRLTISKDNSKSQVFLKLNSL FR3QTDDTATYYC 162 hMC48 VH WGQGTLVTVSS FR4

Antibodies Specific to SSEA4 and Fragment Thereof

One aspect of the present disclosure features the new antibodies thatbind to SSEA-4 and fragments thereof. The anti-SSEA-4 antibody binds toNeu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide)and Neu5Aca2→3Galβ1→3GalNAcβ1→3Galα1(fragment of SSEA-4 hexasaccharide).In some examples, the antibody is capable ofNeu5Aca2→3Galβ1→3GalNAcβ1→3Galβ1. In some examples, the antibody iscapable of Neu5Gca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (an analogueof SSEA-4 hexasaccharide).

In some embodiments, the method enhances ADCC.

In one embodiment, the pharmaceutical composition comprises antibodiesor binding fragments thereof having universal biantennary n-glycanterminated with sialic acid in alpha-2,6-linkage.

In another aspect, the present invention provides methods for treatingand/or reducing the risk for cancer in a subject comprisingadministering to a subject in need thereof a therapeutically effectiveamount of composition as described herein.

The treatment results in reduction of tumor size, elimination ofmalignant cells, prevention of metastasis, prevention of relapse,reduction or killing of disseminated cancer, prolongation of survivaland/or prolongation of time to tumor cancer progression.

In some embodiments, the composition described herein is formulated aninjectable. In some embodiments, the composition is administeredsubcutaneously.

The details of certain embodiments of the invention are set forthherein. Other features, objects, and advantages of the invention will beapparent from the Detailed Description, the Figures, the Examples, andthe Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of optimized universal Fc glycan of therapeuticantibodies.

FIG. 2. General strategy for the preparation of homogeneous antibodywith optimized universal glycan at the Fc region for the improvement ofits therapeutic activity.

FIG. 3. Demonstrates the enhanced anti-viral antibody-dependentcell-mediated cytotoxicity (ADCC) results of anti-influenza virusantibodies.

FIG. 4. Table listing exemplary enhanced ADCC activities of anti-CD20GAbs as compared to Rituximab.

FIG. 5. Six anti-CD20 GAbs

FIGS. 6A and 6B. FIG. 6A is top of table, FIG. 6B is bottom of table.Table lists exemplary FcγRIIIA binding of anti-CD20 GAbs and Rituximab.FcγRIIIA binding may be measured using assays known in the art.Exemplary assays are described in the examples. The Fc receptor bindingmay be determined as the relative ratio of anti-CD20 GAb vs Rituximab.Fc receptor binding in exemplary embodiments is increased by at least1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 15-fold or 20-fold, 30-fold, 40-fold, 50-fold, 100-foldor higher.

FIG. 7. Binding activities of different homogeneous antibodies withdifferent cells with CD20. FIG. 7 shows CDC effects of Rituxan-SCT(Gab101) and Rituxan mono-GlcNAc to Ramos cells.

FIG. 8. Binding activities of different homogeneous antibodies withdifferent cells with CD20. FIG. 8 shows CDC effects of Rituxan-SCT(Gab101) and Rituxan mono-GlcNAc to Raji cells.

FIG. 9. Binding activities of different homogeneous antibodies withdifferent cells with CD20. FIG. 9 shows CDC effects of Rituxan-SCT(Gab101) and Rituxan mono-GlcNAc to SU-DHL-4 cells.

FIG. 10. Depletion of human SU-DHL-4 B cells as analyzed on FACS. Cellswere cultured in the absence or presence of 15% autologous plasma withanti-CD20 Gabs Rituxan-SCT, Rituxan-GlcNAc and Rituximab at differentconcentrations. After wash cells were stained with anti-CD2-PE andanti-CD19-FITC. B cell depletion was analyzed on FACS, based on theCD19+CD2-B cells (FIG. 13).

FIG. 11. Depletion of human Ramos B cells as analyzed on FACS. Cellswere cultured in the absence or presence of 15% autologous plasma withanti-CD20 Gabs Rituxan-SCT, Rituxan-GlcNAc and Rituximab at differentconcentrations. After wash cells were stained with anti-CD2-PE andanti-CD19-FITC. B cell depletion was analyzed on FACS, based on theCD19+CD2-B cells (FIG. 13).

FIG. 12. Depletion of human Raji B cells as analyzed on FACS. Cells werecultured in the absence or presence of 15% autologous plasma withanti-CD20 Gabs Rituxan-SCT, Rituxan-GlcNAc and Rituximab at differentconcentrations. After wash cells were stained with anti-CD2-PE andanti-CD19-FITC. B cell depletion was analyzed on FACS, based on theCD19+CD2-B cells (FIG. 13).

FIG. 13. Depletion of human B cells by different homogeneous antibodies.

FIG. 14. Table listing exemplary enhanced ADCC activities of anti-HER2GAbs as compared to Trastuzumab.

FIG. 15. Table listing exemplary FcγRIIIA binding of anti-HER2 GAbs andRituximab.

FIG. 16A. Solid-based ELISA coating SSEA-4 to determine the bindingactivity of humanized MC41 phage clones

FIG. 16B. Solid-based ELISA coating BSA to determine the bindingactivity of humanized MC41 phage clones

FIG. 17A. To evaluate the binding activity by intact humanized MC41 IgG,intact IgGs of 1st, 2nd, 3rd humanized MC41, and chimeric MC41 (chMC41)are contructed. The ELISA results show that the humanized 2nd and 3rdMC41 could react to SSEA-4 (FIG. 17A) but not to BSA (FIG. 17B) in adose-dependent pattern, same results were observed for chMC41.

FIG. 17B. To evaluate the binding activity by intact humanized MC41 IgG,intact IgGs of 1st, 2nd, 3rd humanized MC41, and chimeric MC41 (chMC41)are contructed. The ELISA results show that the humanized 2nd and 3rdMC41 could react to SSEA-4 (FIG. 17A) but not to BSA (FIG. 17B) in adose-dependent pattern, same results were observed for chMC41.

FIG. 18A and FIG. 18B. FIG. 18A shows the legend for bar graph of FIG.18B. In order to determine the binding specificity of chMC41 and hMC41,glycan array is performed. Results are shown in FIG. 18B. The chimericand humanized MC41 show more specific binding than commercial SSEA4antibody (MC813). They only recognized SSEA4 or glycolyl modified SSEA4.

FIGS. 19A and 19B. FIG. 19A shows the legend for the bar graph of FIG.19B. In order to determine the binding specificity of chMC41 and hMC41,glycan array is performed. Results are shown in FIG. 19B. The chimericand humanized MC41 show more specific binding than commercial SSEA4antibody (MC813). They only recognized SSEA4 or glycolyl modified SSEA4.

FIG. 20A. To investigate the effector function of chMC41 and hMC41, ADCCand CDC assays were performed. HPAC pancreatic cancer cell line was usedto evaluate the ADCC and CDC activities of chMC41, hMC41, positivecontrol MC813 or negative controls NHIgG and NMIgG.

FIG. 20B. To investigate the effector function of chMC41 and hMC41, ADCCand CDC assays were performed. HPAC pancreatic cancer cell line was usedto evaluate the ADCC and CDC activities of chMC41, hMC41, positivecontrol MC813 or negative controls NHIgG and NMIgG.

FIG. 21A and FIG. 21B. To investigate the effector function of chMC41and hMC41, ADCC and CDC assays were performed. HPAC pancreatic cancercell line was used to evaluate the ADCC and CDC activities of chMC41,hMC41, positive control MC813 or negative controls NHIgG and NMIgG. FIG.21A shows cancer cell killing activity through ADCC. FIG. 21B showscancer cell killing activity through CDC.

FIG. 22A. To identify the antibodies that bind to SSEA-4, we usedphage-displayed human naïve scFv library containing 2×1010 members,which was established as described in our previous report (Lu et al.,2011). This library was first removed by Dynabeads-binding phages, andthen SSEA-4-binding phages were selected by SSEA-4-PEG-conjugatedDynabeads. We used two buffer systems, PBS and PBS containing 0.01%Tween20 (PBST0.01), during biopanning. After five rounds of affinityselection, the phage recovery of the fifth round increased by about55-fold and 80-fold, compared to that of the first round in PBS andPBST0.01 system, respectively.

FIG. 22B. To identify the antibodies that bind to SSEA-4, we usedphage-displayed human naïve scFv library containing 2×1010 members,which was established as described in our previous report (Lu et al.,2011). This library was first removed by Dynabeads-binding phages, andthen SSEA-4-binding phages were selected by SSEA-4-PEG-conjugatedDynabeads. We used two buffer systems, PBS and PBS containing 0.01%Tween20 (PBST0.01), during biopanning. After five rounds of affinityselection, the phage recovery of the fifth round increased by about55-fold and 80-fold, compared to that of the first round in PBS andPBST0.01 system, respectively.

FIG. 23A. The phage clones were randomly selected and tested for SSEA-4binding by ELISA

FIG. 23B. The phage clones were randomly selected and tested for SSEA-4binding by ELISA

FIG. 23C. The phage clones were randomly selected and tested for SSEA-4binding by ELISA

FIG. 23D. The phage clones were randomly selected and tested for SSEA-4binding by ELISA

FIG. 24. To examine the specificity and binding affinity of the twophage clones, we performed a comparative ELISA using the same phagetiter to Globo-series glycans including SSEA-4-BSA, Globo H-BSA andSSEA-3-BSA.

FIG. 25A. To establish the fully human antibody (hAb) against SSEA-4, wemolecularly engineered the VH and VL coding sequences of p2-78 scFv intohuman IgG1 backbone, respectively. The anti-SSEA-4 p2-78 hAb wasproduced using FreeStyle 293 expression system and then purified throughthe protein G sepharose column. We examined the purity of antibody bySDS-PAGE analysis with coomassie blue staining

FIG. 25B. ELISA to investigate the binding activity of p2-78 hAb forGlobo-series glycans.

FIG. 26A. Positive control of commercially available IgM antibody,MC631. Glycan array containing 203 different glycans to further confirmthe specificity of p2-78 hAb.

FIG. 26B. Glycans recognized by p2-78 hAb.

FIG. 26C. Glycan array containing 203 different glycans to furtherconfirm the specificity of p2-78 hAb.

FIG. 27A. After alignment of VH and VL variable region of MC48 and MC41with the NCBI IgBLAST or IMGT database, we generated 1st, 2nd, 3rd and4th humanized MC48 sequences and 1st, 2nd and 3rd humanized MC41sequences. We next constructed and generated the phage-displayed scFvformats according to these humanized MC48 and MC41 sequences. Todetermine the binding activity of the humanized MC48 and MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA. We foundthat the 3rd and 4th humanized MC48, and 2nd and 3rd humanized MC41 scFvphages could recognize SSEA-4 in a dose-dependent manner, whereas the1st and 2nd humanized MC48 and 1st MC41 scFv lost the binding activityto SSEA-4. The data showed that the binding affinities of the 4thhumanized MC48, and 3rd humanized MC41 scFv phage clones weremaintained, compared to that of the murine mAbs MC48 or MC41.

FIG. 27B. After alignment of VH and VL variable region of MC48 and MC41with the NCBI IgBLAST or IMGT database, we generated 1st, 2nd, 3rd and4th humanized MC48 sequences and 1st, 2nd and 3rd humanized MC41sequences. We next constructed and generated the phage-displayed scFvformats according to these humanized MC48 and MC41 sequences. Todetermine the binding activity of the humanized MC48 and MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA. We foundthat the 3rd and 4th humanized MC48, and 2nd and 3rd humanized MC41 scFvphages could recognize SSEA-4 in a dose-dependent manner, whereas the1st and 2nd humanized MC48 and 1st MC41 scFv lost the binding activityto SSEA-4. The data showed that the binding affinities of the 4thhumanized MC48, and 3rd humanized MC41 scFv phage clones weremaintained, compared to that of the murine mAbs MC48 or MC41.

FIG. 28A. After alignment of VH and VL variable region of MC48 and MC41with the NCBI IgBLAST or IMGT database, we generated 1st, 2nd, 3rd and4th humanized MC48 sequences and 1st, 2nd and 3rd humanized MC41sequences. We next constructed and generated the phage-displayed scFvformats according to these humanized MC48 and MC41 sequences. Todetermine the binding activity of the humanized MC48 and MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA. We foundthat the 3rd and 4th humanized MC48, and 2nd and 3rd humanized MC41 scFvphages could recognize SSEA-4 in a dose-dependent manner, whereas the1st and 2nd humanized MC48 and 1st MC41 scFv lost the binding activityto SSEA-4. The data showed that the binding affinities of the 4thhumanized MC48, and 3rd humanized MC41 scFv phage clones weremaintained, compared to that of the murine mAbs MC48 or MC41.

FIG. 28B. After alignment of VH and VL variable region of MC48 and MC41with the NCBI IgBLAST or IMGT database, we generated 1st, 2nd, 3rd and4th humanized MC48 sequences and 1st, 2nd and 3rd humanized MC41sequences. We next constructed and generated the phage-displayed scFvformats according to these humanized MC48 and MC41 sequences. Todetermine the binding activity of the humanized MC48 and MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA. We foundthat the 3rd and 4th humanized MC48, and 2nd and 3rd humanized MC41 scFvphages could recognize SSEA-4 in a dose-dependent manner, whereas the1st and 2nd humanized MC48 and 1st MC41 scFv lost the binding activityto SSEA-4. The data showed that the binding affinities of the 4thhumanized MC48, and 3rd humanized MC41 scFv phage clones weremaintained, compared to that of the murine mAbs MC48 or MC41.

FIG. 29A and FIG. 29B. After alignment of VH and VL variable region ofMC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated 1st,2nd, 3rd and 4th humanized MC48 sequences and 1st, 2nd and 3rd humanizedMC41 sequences. We next constructed and generated the phage-displayedscFv formats according to these humanized MC48 and MC41 sequences. Todetermine the binding activity of the humanized MC48 and MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA. We foundthat the 3rd and 4th humanized MC48, and 2nd and 3rd humanized MC41 scFvphages could recognize SSEA-4 in a dose-dependent manner, whereas the1st and 2nd humanized MC48 and 1st MC41 scFv lost the binding activityto SSEA-4. The data showed that the binding affinities of the 4thhumanized MC48, and 3rd humanized MC41 scFv phage clones weremaintained, compared to that of the murine mAbs MC48 or MC41.

FIG. 29B. After alignment of VH and VL variable region of MC48 and MC41with the NCBI IgBLAST or IMGT database, we generated 1st, 2nd, 3rd and4th humanized MC48 sequences and 1st, 2nd and 3rd humanized MC41sequences. We next constructed and generated the phage-displayed scFvformats according to these humanized MC48 and MC41 sequences. Todetermine the binding activity of the humanized MC48 and MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA. We foundthat the 3rd and 4th humanized MC48, and 2nd and 3rd humanized MC41 scFvphages could recognize SSEA-4 in a dose-dependent manner, whereas the1st and 2nd humanized MC48 and 1st MC41 scFv lost the binding activityto SSEA-4. The data showed that the binding affinities of the 4thhumanized MC48, and 3rd humanized MC41 scFv phage clones weremaintained, compared to that of the murine mAbs MC48 or MC41.

FIGS. 30A and 30B. To evaluate the binding activity by intact humanizedMC41 IgG, we constructed intact IgGs of 1st, 2nd, 3rd humanized MC41 andchimeric MC41 (chMC41). The ELISA results showed that the humanized 2ndand 3rd MC41 could react to SSEA-4 (FIG. 30A) but not to BSA (FIG. 30B)in a dose-dependent pattern, same results were observed for chMC41.

FIG. 31A and FIG. 31B. In order to determine the binding specificity ofchMC41 and hMC41, glycan array was performed. The chimeric and humanizedMC41 showed more specific binding than commercial SSEA4 antibody(MC813). They only recognized SSEA4 or glycolyl modified SSEA4. FIG. 31Ashows the glycans that were recognized and FIG. 31B shows the arrayresults.

FIG. 32A and FIG. 32B. In order to determine the binding specificity ofchMC41 and hMC41, glycan array was performed. The chimeric and humanizedMC41 showed more specific binding than commercial SSEA4 antibody(MC813). They only recognized SSEA4 or glycolyl modified SSEA4. FIG. 32Ashows the glycans that were recognized and FIG. 32B shows the arrayresults.

FIG. 33A and FIG. 33B. To investigate the effector function of hMC48,chMC41 and hMC41, ADCC and CDC assays were performed. HPAC, BxPC3 andPL45 pancreatic cancer cell lines were used to evaluate the ADCC and CDCactivities at the concentration of 10 μg/ml for hMC48 or NHIgG.

FIG. 34A. HPAC cells were treated with chMC41, hMC41, positive controlMC813 or negative control NHIgG.

FIG. 34B. HPAC cells were treated with chMC41, hMC41, positive controlMC813 or negative control NHIgG.

FIGS. 35A and 35B. The data showed that the effector function of hMC41and chMC41 was superior to that of hMC48. Interestingly, the humanizedMC41 not only maintain its original activity, but it also showedstronger cancer cell killing activity than MC813 through ADCC and CDC.

FIG. 36. The binding abilities of hMC41 and hMC48 to SSEA-4 wereexamined by ELISA. The result showed that the binding of hMC41 to SSEA-4was much better than hMC48. The humanized MC41 has a higher bindingmaximum and a smaller Kd (0.2 μg/ml and 4.6 μg/ml for hMC41 and hMC48,respectively) value as compared to hMC48.

DETAILED DESCRIPTIONS Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987. Moreover, exemplary glycan andantibody methodologies are described in Wong et al, US20100136042,US20090317837, and US20140051127, the disclosures of each of which arehereby incorporated by reference.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆” is intended toencompass C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning(1984); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology,Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (ColdSpring Harbor Laboratory Press, 1988); and Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

As used herein, the term “glycan” refers to a polysaccharide, oroligosaccharide. Glycan is also used herein to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid,glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or aproteoglycan. Glycans usually consist solely of O-glycosidic linkagesbetween monosaccharides. For example, cellulose is a glycan (or morespecifically a glucan) composed of β-1,4-linked D-glucose, and chitin isa glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can behomo or heteropolymers of monosaccharide residues, and can be linear orbranched. Glycans can be found attached to proteins as in glycoproteinsand proteoglycans. They are generally found on the exterior surface ofcells. O- and N-linked glycans are very common in eukaryotes but mayalso be found, although less commonly, in prokaryotes. N-Linked glycansare found attached to the R-group nitrogen (N) of asparagine in thesequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is anyamino acid except praline.

As used herein, the term “epitope” is defined as the parts of an antigenmolecule which contact the antigen binding site of an antibody or a Tcell receptor.

As used herein, the term “Flow cytometry” or “FACS” means a techniquefor examining the physical and chemical properties of particles or cellssuspended in a stream of fluid, through optical and electronic detectiondevices.

A non-naturally occurring or an “isolated” antibody is one which hasbeen identified and separated and/or recovered from a component of itsnative environment. Contaminant components of its native environment arematerials which would interfere with research, diagnostic or therapeuticuses for the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In one embodiment, theantibody will be purified (1) to greater than 95% by weight of antibodyas determined by, for example, the Lowry method, and in some embodimentsmore than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of, forexample, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using, for example, Coomassieblue or silver stain. Isolated antibody includes the antibody in situwithin recombinant cells since at least one component of the antibody'snatural environment will not be present. Ordinarily, however, isolatedantibody will be prepared by at least one purification step.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise an antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

Identity or homology with respect to a specified amino acid sequence ofthis invention is defined herein as the percentage of amino acidresidues in a candidate sequence that are identical with the specifiedresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology, and not consideringany conservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal or internal extensions, deletions, or insertionsinto the specified sequence shall be construed as affecting homology.All sequence alignments called for in this invention are such maximalhomology alignments. Generally, the nucleic acid sequence homologybetween the polynucleotides, oligonucleotides, and fragments of theinvention and a nucleic acid sequence of interest will be at least 80%>,and more typically with preferably increasing homologies of at least85%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%. Twoamino acid sequences are homologous if there is a partial or completeidentity between their sequences.

The term “globoseries-related disorder” refers to or describes adisorder that is typically characterized by or contributed to byaberrant functioning or presentation of the pathway. Examples of suchdisorders include, but are not limited to, hyperproliferative diseases,including cancer.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing or decreasing inflammation and/or tissue/organdamage, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning(1984); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology,Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (ColdSpring Harbor Laboratory Press, 1988); and Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

As used herein, the term “glycan” refers to a polysaccharide, oroligosaccharide. Glycan is also used herein to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid,glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or aproteoglycan. Glycans usually consist solely of O-glycosidic linkagesbetween monosaccharides. For example, cellulose is a glycan (or morespecifically a glucan) composed of β-1,4-linked D-glucose, and chitin isa glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can behomo or heteropolymers of monosaccharide residues, and can be linear orbranched. Glycans can be found attached to proteins as in glycoproteinsand proteoglycans. They are generally found on the exterior surface ofcells. O- and N-linked glycans are very common in eukaryotes but mayalso be found, although less commonly, in prokaryotes. N-Linked glycansare found attached to the R-group nitrogen (N) of asparagine in thesequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is anyamino acid except praline.

As used herein, the term “antigen” is defined as any substance capableof eliciting an immune response.

As used herein, the term “immunogenicity” refers to the ability of animmunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “CD1d” refers to a member of the CD1 (clusterof differentiation 1) family of glycoproteins expressed on the surfaceof various human antigen-presenting cells. CD1d presented lipid antigensactivate natural killer T cells. CD1d has a deep antigen-binding grooveinto which glycolipid antigens bind. CD1d molecules expressed ondendritic cells can bind and present glycolipids, including alpha-GalCeranalogs such as C34.

As used herein, the term “epitope” is defined as the parts of an antigenmolecule which contact the antigen binding site of an antibody or a Tcell receptor.

As used herein, the term “vaccine” refers to a preparation that containsan antigen, consisting of whole disease-causing organisms (killed orweakened) or components of such organisms, such as proteins, peptides,or polysaccharides, that is used to confer immunity against the diseasethat the organisms cause. Vaccine preparations can be natural, syntheticor derived by recombinant DNA technology.

As used herein, the term “antigen specific” refers to a property of acell population such that supply of a particular antigen, or a fragmentof the antigen, results in specific cell proliferation.

As used herein, the term “specifically binding,” refers to theinteraction between binding pairs (e.g., an antibody and an antigen). Invarious instances, specifically binding can be embodied by an affinityconstant of about 10-6 moles/liter, about 10-7 moles/liter, or about10-8 moles/liter, or less.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In one embodiment, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined by,for example, the Lowry method, and in some embodiments more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

The phrase “substantially similar,” “substantially the same”,“equivalent”, or “substantially equivalent”, as used herein, denotes asufficiently high degree of similarity between two numeric values (forexample, one associated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said values (e.g., Kd values,anti-viral effects, etc.). The difference between said two values is,for example, less than about 50%, less than about 40%, less than about30%, less than about 20%, and/or less than about 10% as a function ofthe value for the reference/comparator molecule.

The phrase “substantially reduced,” or “substantially different”, asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (MA) performed with theFab version of an antibody of interest and its antigen as described bythe following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(125I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [125I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. In each experiment, a spot wasactivated and ethanolamine blocked without immobilizing protein, to beused for reference subtraction. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (kon) and dissociation rates (koff) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen, Y.,et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds 106 M−1s−1 by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with astirred cuvette.

An “on-rate” or “rate of association” or “association rate” or “kon”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (kon) and dissociation rates (koff) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgram. The equilibrium dissociationconstant (Kd) was calculated as the ratio koff/kon. See, e.g., Chen, Y.,et al., (1999) J. Mol Biol 293:865-881. However, if the on-rate exceeds106 M−1 s−1 by the surface plasmon resonance assay above, then theon-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stirred cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3 terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R,P(O)OR′, CO or CH2 (“formacetal”), in which each R or R is independentlyH or substituted or unsubstituted alkyl (1-20 C) optionally containingan ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl oraraldyl. Not all linkages in a polynucleotide need be identical. Thepreceding description applies to all polynucleotides referred to herein,including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, monovalent,multivalent antibodies, multi specific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be chimeric, human, humanized and/oraffinity matured.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of heavy or light chain of the antibody. Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably, to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise an antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts or comprising only homogeneous glycoform profile (havingonly a single glycan or single glycan profile on a glycoantibody in apopulation). Examples of homogeneous antibody composition to enhance theeffector functions by using the 2,3- and 2,6-sialyl and defucosylatedcomplex bi-antennary glycans at the Fc-297 position are described inU.S. Ser. No. 12/959,351. Thus, the modifier “monoclonal” indicates thecharacter of the antibody as not being a mixture of discrete antibodies.Such monoclonal antibody typically includes an antibody comprising apolypeptide sequence that binds a target, wherein the target-bindingpolypeptide sequence was obtained by a process that includes theselection of a single target binding polypeptide sequence from aplurality of polypeptide sequences. For example, the selection processcan be the selection of a unique clone from a plurality of clones, suchas a pool of hybridoma clones, phage clones or recombinant DNA clones.It should be understood that the selected target binding sequence can befurther altered, for example, to improve affinity for the target, tohumanize the target binding sequence, to improve its production in cellculture, to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered targetbinding sequence is also a monoclonal antibody of this invention. Incontrast to polyclonal antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody of a monoclonal antibody preparation isdirected against a single determinant on an antigen. In addition totheir specificity, the monoclonal antibody preparations are advantageousin that they are typically uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by a variety oftechniques, including, for example, the hybridoma method (e.g., Kohleret al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerlinget al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681(Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567), phage display technologies (See, e.g., Clackson et al.,Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods284(1-2): 119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovitset al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992);Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar,Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact

L1 L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

H1 H31-H35B H26-H35B H26-H32 H30-H35B

(Kabat Numbering)

H1 H31-H35 H26-H35 H26-H32 H30-H35

(Chothia Numbering)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 or 49-56 (L2) and 89-97 or89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102,94-102, or 95-102 (H3) in the VH. The variable domain residues arenumbered according to Kabat et al., supra, for each of thesedefinitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or HVR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO93/1161; and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies are produced by procedures known inthe art. Marks et al. Bio/Technology 10:779-783 (1992) describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with anantibody of the invention. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include cancer.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, leukemia and other lymphoproliferative disorders, andvarious types of head and neck cancer.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing or decreasing inflammation and/or tissue/organdamage, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder.

An “individual” or a “subject” is a vertebrate. In certain embodiments,the vertebrate is a mammal. Mammals include, but are not limited to,farm animals (such as cows), sport animals, pets (such as cats, dogs,and horses), primates, mice and rats. In certain embodiments, thevertebrate is a human.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. In certainembodiments, the mammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the substance/molecule are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolyticenzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammaII and calicheamicin omegaII (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Pharmaceutical Formulations

The pharmaceutical composition is administered in a manner compatiblewith the dosage formulation, and in an amount that is therapeuticallyeffective, protective and therapeutic. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art. Suitable regimes for initial administrationand booster doses are also variable, but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and variesaccording to the size of the host.

Methods of making monoclonal and polyclonal antibodies and fragmentsthereof in animals (e.g., mouse, rabbit, goat, sheep, or horse) are wellknown in the art. See, for example, Harlow and Lane, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term“antibody” includes intact immunoglobulin molecules as well as fragmentsthereof, such as Fab, F(ab′)2, Fv, scFv (single chain antibody), and dAb(domain antibody; Ward, et. al. (1989) Nature, 341, 544).

The compositions disclosed herein can be included in a pharmaceuticalcomposition together with additional active agents, carriers, vehicles,excipients, or auxiliary agents identifiable by a person skilled in theart upon reading of the present disclosure.

The pharmaceutical compositions preferably comprise at least onepharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the compositions disclosed herein form the “activecompound,” also referred to as the “active agent.” As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates, or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Clinical Applications

The present invention provides selected and directed optimizedglycoantibodies useful for the treatment of a proliferative disease suchas cancer (e.g. lung cancer, large bowel cancer, pancreas cancer,biliary tract cancer, or endometrial cancer), benign neoplasm, orangiogenesis in a subject.

The compositions described herein can also be used in both cancertreatment and diagnosis. Methods of making monoclonal and polyclonalantibodies and fragments thereof in human and/or animals (e.g., mouse,rabbit, goat, sheep, or horse) are well known in the art. See, forexample, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. The term “antibody” includes intactimmunoglobulin molecules as well as fragments thereof, such as Fab,F(ab′).sub.2, Fv, scFv (single chain antibody), and dAb (domainantibody; Ward, et. al. (1989) Nature, 341, 544).

These compositions may further comprise suitable carriers, such aspharmaceutically acceptable excipients including buffers, which are wellknown in the art.

Non naturally occurring and or isolated antibodies and polynucleotidesare also provided. In certain embodiments, the isolated antibodies andpolynucleotides are substantially pure.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naïverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naïve libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, the library diversity is maximized byusing PCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naïve VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 1012 clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (Kd-1 of about 10-8 M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

Screening of the libraries can be accomplished by any art-knowntechnique. Targets can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other art-known method for panning phage displaylibraries.

The phages bound to the solid phase are washed and then eluted by acid,e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J.Mol. Biol., 222: 581-597 (1991), or by SSEA-3/SSEA-4/GLOBO H antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting SSEA-3/SSEA-4/GLOBO H, rare high affinityphage could be competed out. To retain all the higher affinity mutants,phages can be incubated with excess biotinylated SSEA-3/SSEA-4/GLOBO H,but with the biotinylated SSEA-3/SSEA-4/GLOBO H at a concentration oflower molarity than the target molar affinity constant forSSEA-3/SSEA-4/GLOBO H. The high affinity-binding phages can then becaptured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics.

DNA encoding the Fv clones of the invention is readily isolated andsequenced using conventional procedures (e.g. by using oligonucleotideprimers designed to specifically amplify the heavy and light chaincoding regions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In one embodiment, a Fvclone derived from human variable DNA is fused to human constant regionDNA to form coding sequence(s) for all human, full or partial lengthheavy and/or light chains.

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (Kd-1 of about 106 to 107 M−1), but affinitymaturation can also be mimicked in vitro by constructing and reselectingfrom secondary libraries as described in Winter et al. (1994), supra.For example, mutation can be introduced at random in vitro by usingerror-prone polymerase (reported in Leung et al., Technique, 1: 11-15(1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896(1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). Additionally, affinity maturation can be performed byrandomly mutating one or more CDRs, e.g. using PCR with primers carryingrandom sequence spanning the CDR of interest, in selected individual Fvclones and screening for higher affinity clones. WO 9607754 (published14 Mar. 1996) described a method for inducing mutagenesis in acomplementarity determining region of an immunoglobulin light chain tocreate a library of light chain genes. Another effective approach is torecombine the VH or VL domains selected by phage display withrepertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10-9 M range.

Other methods of generating and assessing the affinity of antibodies arewell known in the art and are described, e.g., in Kohler et al., Nature256: 495 (1975); U.S. Pat. No. 4,816,567; Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986; Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987; Munson et al., Anal. Biochem., 107:220 (1980); Engels etal., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989); Abrahmsen et al.,EMBO J., 4: 3901 (1985); Methods in Enzymology, vol. 44 (1976); Morrisonet al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).

General Methods

Generation of antibodies can be achieved using routine skills in theart, including those described herein, such as the hybridoma techniqueand screening of phage displayed libraries of binder molecules. Thesemethods are well-established in the art.

Briefly, antibodies of the invention can be made by using combinatoriallibraries to screen for synthetic antibody clones with the desiredactivity or activities. In principle, synthetic antibody clones areselected by screening phage libraries containing phage that displayvarious fragments of antibody variable region (Fv) fused to phage coatprotein. Such phage libraries are panned by affinity chromatographyagainst the desired antigen. Clones expressing Fv fragments capable ofbinding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clonesare then eluted from the antigen, and can be further enriched byadditional cycles of antigen adsorption/elution. Any of the antibodiesof the invention can be obtained by designing a suitable antigenscreening procedure to select for the phage clone of interest followedby construction of a full length antibody clone using the Fv sequencesfrom the phage clone of interest and suitable constant region (Fc)sequences described in Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, NIH Publication 91-3242, BethesdaMd. (1991), vols. 1-3.

Monoclonal antibodies can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The monoclonal antibodies of the invention can be made using a varietyof methods known in the art, including the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or alternativelythey may be made by recombinant DNA methods (e.g., U.S. Pat. No.4,816,567).

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Host cells include,but are not limited to, cells of either prokaryotic or eukaryotic(generally mammalian) origin. It will be appreciated that constantregions of any isotype can be used for this purpose, including IgG, IgM,IgA, IgD, and IgE constant regions, and that such constant regions canbe obtained from any human or animal species.

Generating Antibodies Using Prokaryotic Host Cells

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, growth occurs at a temperature rangeincluding, but not limited to, about 20° C. to about 39° C., about 25°C. to about 37° C., and at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH can be from about 6.8 to about 7.4, orabout 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In one embodiment, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, for example about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (a common carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected generally is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II (e.g., primate metallothionein genes), adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene may first beidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR.Appropriate host cells when wild-type DHFR is employed include, forexample, the Chinese hamster ovary (CHO) cell line deficient in DHFRactivity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. Virtually all eukaryotic genes havean AT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellscan be controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human (3-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding an antibody polypeptide of the inventionby higher eukaryotes can often be increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5 or 3′ to theantibody polypeptide-encoding sequence, but is generally located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TM cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are generally removed, forexample, by centrifugation or ultrafiltration. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a generally acceptable purification technique. The suitability ofaffinity reagents such as protein A as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is present inthe antibody. Protein A can be used to purify antibodies that are basedon human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification steps, as necessary, for example by low pH hydrophobicinteraction chromatography using an elution buffer at a pH between about2.5-4.5, generally performed at low salt concentrations (e.g., fromabout 0-0.25M salt).

It should be noted that, in general, techniques and methodologies forpreparing antibodies for use in research, testing and clinical use arewell-established in the art, consistent with the above and/or as deemedappropriate by one skilled in the art for the particular antibody ofinterest.

Activity Assays

Antibodies of the invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

Purified antibodies can be further characterized by a series of assaysincluding, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

Where necessary, antibodies are analyzed for their biological activity.In some embodiments, antibodies of the invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays.

In one embodiment, the invention contemplates an altered antibody thatpossesses some but not all effector functions, which make it a desirablecandidate for many applications in which the half life of the antibodyin vivo is important yet certain effector functions (such as complementand ADCC) are unnecessary or deleterious. In certain embodiments, the Fcactivities of the antibody are measured to ensure that only the desiredproperties are maintained. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., in aanimal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1998). C1q binding assays may also be carried out to confirmthat the antibody is unable to bind C1q and hence lacks CDC activity. Toassess complement activation, a CDC assay, e.g. as described inGazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may beperformed. FcRn binding and in vivo clearance/half life determinationscan also be performed using methods known in the art.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)2 fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab) 2 fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)2 fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

Any of the antibodies described herein can be a full length antibody oran antigen-binding fragment thereof. In some examples, the antigenbinding fragment is a Fab fragment, a F(ab′)2 fragment, or asingle-chain Fv fragment. In some examples, the antigen binding fragmentis a Fab fragment, a F(ab′)2 fragment, or a single-chain Fv fragment. Insome examples, the isolated antibody is a human antibody, a humanizedantibody, a chimeric antibody, or a single-chain antibody.

Any of the antibodies described herein has one or more characteristicsof:

a) is a recombinant antibody, a monoclonal antibody, a chimericantibody, a humanized antibody, a human antibody, an antibody fragment,a bispecific antibody, a monospecific antibody, a monovalent antibody,an IgG1 antibody, an IgG2 antibody, or derivative of an antibody; b) isa human, murine, humanized, or chimeric antibody, antigen-bindingfragment, or derivative of an antibody; c) is a single-chain antibodyfragment, a multibody, a Fab fragment, and/or an immunoglobulin of theIgG, IgM, IgA, IgE, IgD isotypes and/or subclasses thereof; d) has oneor more of the following characteristics: (i) mediates ADCC and/or CDCof cancer cells; (ii) induces and/or promotes apoptosis of cancer cells;(iii) inhibits proliferation of target cells of cancer cells; (iv)induces and/or promotes phagocytosis of cancer cells; and/or (v) inducesand/or promotes the release of cytotoxic agents; e) specifically bindsthe tumor-associated carbohydrate antigen, which is a tumor-specificcarbohydrate antigen; f) does not bind an antigen expressed onnon-cancer cells, non-tumor cells, benign cancer cells and/or benigntumor cells; and/or g) specifically binds a tumor-associatedcarbohydrate antigen expressed on cancer stem cells and on normal cancercells.

Preferably the binding of the antibodies to their respective antigens isspecific. The term “specific” is generally used to refer to thesituation in which one member of a binding pair will not show anysignificant binding to molecules other than its specific binding partner(s) and e.g. has less than about 30%, preferably 20%, 10%, or 1%cross-reactivity with any other molecule other than those specifiedherein.

The antibodies are suitable bind to its target epitopes with a highaffinity (low KD value), and preferably KD is in the nanomolar range orlower. Affinity can be measured by methods known in the art, such as,for example; surface plasmon resonance.

Exemplary Antibody Preparation

Exemplary Antibodies capable of binding to the Globo H epitopes andSSEA-4 epitopes described herein can be made by any method known in theart. See, for example, Harlow and Lane, (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York.

Immunization of Host Animals and Hybridoma Technology

Exemplary Polyclonal antibodies against the anti-Globo Hand anti-SSEA-4antibodies may be prepared by collecting blood from the immunized mammalexamined for the increase of desired antibodies in the serum, and byseparating serum from the blood by any conventional method. Polyclonalantibodies include serum containing the polyclonal antibodies, as wellas the fraction containing the polyclonal antibodies may be isolatedfrom the serum.

Polyclonal antibodies are generally raised in host animals (e.g.,rabbit, mouse, horse, or goat) by multiple subcutaneous (sc) orintraperitoneal (ip) injections of the relevant antigen and an adjuvant.It may be useful to conjugate the relevant antigen to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, etc.

Any mammalian animal may be immunized with the antigen for producing thedesired antibodies. In general, animals of Rodentia, Lagomorpha, orPrimates can be used. Animals of Rodentia include, for example, mouse,rat, and hamster. Animals of Lagomorpha include, for example, rabbit.Animals of Primates include, for example, a monkey of Catarrhini (oldworld monkey) such as Macaca fascicularis, rhesus monkey, baboon, andchimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for immunization of mammals. More specifically, antigensmay be diluted and suspended in an appropriate amount of phosphatebuffered saline (PBS), physiological saline, etc. If desired, theantigen suspension may be mixed with an appropriate amount of a standardadjuvant, such as Freund's complete adjuvant, made into emulsion, andthen administered to mammalian animals. Animals are immunized againstthe antigen, immunogenic conjugates, or derivatives by combining 1 mg or1 μg of the peptide or conjugate (for rabbits or mice, respectively)with 3 volumes of Freund's incomplete adjuvant.

Animals can be boosted until the titer plateaus by severaladministrations of antigen mixed with an appropriately amount ofFreund's incomplete adjuvant every 4 to 21 days. Animals are boostedwith ⅕ to 1/10 the original amount of peptide or conjugate in Freund'scomplete adjuvant by subcutaneous injection at multiple sites. Seven to14 days later the animals are bled and the serum is assayed for antibodytiter. An appropriate carrier may also be used for immunization. Afterimmunization as above, serum is examined by a standard method for anincrease in the amount of desired antibodies. Preferably, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Over the past two to three decades, a number of methodologies have beendeveloped to prepare chimeric, humanized or human antibodies for humanin-vivo therapeutic applications. The most used and proven methodologyis to prepare mouse mAbs using hybridoma methodology and then tohumanize the mAbs by converting the framework regions of the V_(H) andV_(L) domains and constant domains of the mAbs into most homologoushuman framework regions of human V_(H) and V_(L) domains and constantregions of a desirable human γ immunoglobulin isotype and subclass. ManymAbs, such as Xolair, used clinically are humanized mAbs of human γ1, κisotype and subclass and prepared using this methodology.

In some embodiments, antibodies can be made by the conventionalhybridoma technology. Kohler et al., Nature, 256:495 (1975). In thehybridoma method, a mouse or other appropriate host animal, such as ahamster or rabbit, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammalians,and more preferably myeloma cells having an acquired property for theselection of fused cells by drugs.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre et al.,Methods Enzymol. 73:3-46, 1981). Lymphocytes are fused with myelomacells using a suitable fusing agent, such as polyethylene glycol, toform a hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). Resulting hybridomasobtained by the cell fusion may be selected by cultivating them in astandard selection medium, such as HAT medium (hypoxanthine,aminopterin, and thymidine containing medium). The cell culture istypically continued in the HAT medium for several days to several weeks,the time being sufficient to allow all the other cells, with theexception of the desired hybridoma (non-fused cells), to die. Then, thestandard limiting dilution is performed to screen and clone a hybridomacell producing the desired antibody.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay. Measurement of absorbance in enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (MA), and/orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, protein of the invention is appliedto the plate, and then a sample containing a desired antibody, such asculture supernatant of antibody producing cells or purified antibodies,is applied. Then, a secondary antibody that recognizes the primaryantibody and is labeled with an enzyme, such as alkaline phosphatase, isapplied, and the plate is incubated. Next, after washing, an enzymesubstrate, such as p-nitrophenyl phosphate, is added to the plate, andthe absorbance is measured to evaluate the antigen binding activity ofthe sample. A fragment of the protein, such as a C-terminal orN-terminal fragment may be used in this method. BIAcore (Pharmacia) maybe used to evaluate the activity of the antibody according to thepresent invention. The binding affinity of the monoclonal antibody can,for example, be determined by the Scatchard analysis of Munson et al.,Anal. Biochem., 107:220 (1980).

Applying any of the conventional methods, including those describedabove, hybridoma cells producing antibodies that bind to epitopesdescribed herein can be identified and selected for furthercharacterization.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. The monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

In addition, the hybridoma cells may be grown in vivo as ascites tumorsin an animal. For example, the obtained hybridomas can be subsequentlytransplanted into the abdominal cavity of a mouse and the ascites areharvested.

The obtained monoclonal antibodies can be purified by, for example,ammonium sulfate precipitation, a protein A or protein G column, DEAEion exchange chromatography, or an affinity column to which the proteinof the present invention is coupled. The antibody of the presentinvention can be used not only for purification and detection of theprotein of the present invention, but also as a candidate for agonistsand antagonists of the protein of the present invention. In addition,this antibody can be applied to the antibody treatment for diseasesrelated to the protein of the present invention.

Recombinant Technology

The monoclonal antibodies thus obtained can be also recombinantlyprepared using genetic engineering techniques (see, for example,Borrebaeck C. A. K. and Larrick J. W. Therapeutic Monoclonal Antibodies,published in the United Kingdom by MacMillan Publishers LTD, 1990). ADNA encoding an antibody may be cloned from an immune cell, such as ahybridoma or an immunized lymphocyte producing the antibody, insertedinto an appropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

When the obtained antibody is to be administered to the human body(antibody treatment), a human antibody or a humanized antibody ispreferable for reducing immunogenicity. For example, transgenic animalshaving a repertory of human antibody genes may be immunized with anantigen selected from a protein, protein expressing cells, or theirlysates. Antibody producing cells are then collected from the animalsand fused with myeloma cells to obtain hybridoma, from which humanantibodies against the protein can be prepared. Alternatively, an immunecell, such as an immunized lymphocyte, producing antibodies may beimmortalized by an oncogene and used for preparing monoclonalantibodies.

DNA encoding the monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Pluckthun, Immunol. Rev., 130:151-188 (1992).

DNAs encoding the antibodies produced by the hybridoma cells describedabove can be genetically modified, via routine technology, to producegenetically engineered antibodies. Genetically engineered antibodies,such as humanized antibodies, chimeric antibodies, single-chainantibodies, and bi-specific antibodies, can be produced via, e.g.,conventional recombinant technology. The DNA can then be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. In that manner,genetically engineered antibodies, such as “chimeric” or “hybrid”antibodies; can be prepared that have the binding specificity of atarget antigen.

Techniques developed for the production of “chimeric antibodies” arewell known in the art. See, e.g., Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314:452.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i. e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

Any of the nucleic acid encoding the anti-Globo Hand anti-SSEA-4antibodies described herein (including heavy chain, light chain, orboth), vectors such as expression vectors comprising one or more of thenucleic acids, and host cells comprising one or more of the vectors arealso within the scope of the present disclosure. In some examples, avector comprising a nucleic acid comprising a nucleotide sequenceencoding either the heavy chain variable region or the light chainvariable region of an anti-Globo H antibody as described herein. In someexamples, a vector comprising a nucleic acid comprising a nucleotidesequence encoding either the heavy chain variable region or the lightchain variable region of an anti-SSEA-4 antibody as described herein. Inother examples, the vector comprises nucleotide sequences encoding boththe heavy chain variable region and the light chain variable region, theexpression of which can be controlled by a single promoter or twoseparate promoters. Also provided here are methods for producing any ofthe anti-Globo Hand anti-SSEA-4 antibodies as described herein, e.g.,via the recombinant technology described in this section.

Other Technology for Preparing Antibodies

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse® fromAmgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.). In another alternative, antibodies maybe made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, thephage display technology (McCafferty et al., (1990) Nature 348:552-553)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments.

Alternatively, the anti-Globo Hand anti-SSEA-4 antibodies describedherein can be isolated from antibody phage libraries (e.g., single-chainantibody phage libraries) generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol Biol., 222:581-597 (1991).Subsequent publications describe the production of high affinity (nMrange) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

Antibodies obtained as described herein may be purified to homogeneity.For example, the separation and purification of the antibody can beperformed according to separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by the appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing, and others (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988), but are not limited thereto. The concentration of theantibodies obtained as above may be determined by the measurement ofabsorbance, Enzyme-linked immunosorbent assay (ELISA), or so on.Exemplary chromatography, with the exception of affinity includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography, andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1996). The chromatographic procedures can becarried out by liquid-phase chromatography, such as HPLC, FPLC.

The antibodies can be characterized using methods well known in the art.For example, one method is to identify the epitope to which the antigenbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. The epitope can be a linear epitope, i.e., containedin a single stretch of amino acids, or a conformational epitope formedby a three-dimensional interaction of amino acids that may notnecessarily be contained in a single stretch (primary structure linearsequence). Peptides of varying lengths (e.g., at least 4-6 amino acidslong) can be isolated or synthesized (e.g., recombinantly) and used forbinding assays with an antibody. In another example, the epitope towhich the antibody binds can be determined in a systematic screening byusing overlapping peptides derived from the target antigen sequence anddetermining binding by the antibody. According to the gene fragmentexpression assays, the open reading frame encoding the target antigen isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of the antigen with the antibodyto be tested is determined. The gene fragments may, for example, beproduced by PCR and then transcribed and translated into protein invitro, in the presence of radioactive amino acids. The binding of theantibody to the radioactively labeled antigen fragments is thendetermined by immunoprecipitation and gel electrophoresis. Certainepitopes can also be identified by using large libraries of randompeptide sequences displayed on the surface of phage particles (phagelibraries). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays.

In an additional example, mutagenesis of an antigen binding domain,domain swapping experiments and alanine scanning mutagenesis can beperformed to identify residues required, sufficient, and/or necessaryfor epitope binding. For example, domain swapping experiments can beperformed using a mutant of a target antigen in which various residuesin the binding epitope for the candidate antibody have been replaced(swapped) with sequences from a closely related, but antigenicallydistinct protein (such as another member of the neurotrophin proteinfamily). By assessing binding of the antibody to the mutant targetprotein, the importance of the particular antigen fragment to antibodybinding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope (e.g., the MC45 antibody describedherein) as the other antibodies. Competition assays are well known tothose of skill in the art.

Additional Aspects of Exemplary suitable General Antibody ProductionMethods

Methods of making monoclonal and polyclonal antibodies and fragmentsthereof in animals (e.g., mouse, rabbit, goat, sheep, or horse) are wellknown in the art. See, for example, Harlow and Lane, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term“antibody” includes intact immunoglobulin molecules as well as fragmentsthereof, such as Fab, F(ab′)2, Fv, scFv (single chain antibody), and dAb(domain antibody; Ward, et. al. (1989) Nature, 341, 544).

The compositions disclosed herein can be included in a pharmaceuticalcomposition together with additional active agents, carriers, vehicles,excipients, or auxiliary agents identifiable by a person skilled in theart upon reading of the present disclosure.

The pharmaceutical compositions preferably comprise at least onepharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the compositions disclosed herein form the “activecompound,” also referred to as the “active agent.” As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates, or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Compositions comprising at least one anti-SSEA-3/SSEA-4/Globo H antibodyor at least one polynucleotide comprising sequences encoding ananti-SSEA-3/SSEA-4/Globo H antibody are provided. In certainembodiments, a composition may be a pharmaceutical composition. As usedherein, compositions comprise one or more antibodies that bind to one ormore SSEA-3/SSEA-4/Globo H and/or one or more polynucleotides comprisingsequences encoding one or more antibodies that bind to one or moreSSEA-3/SSEA-4/Globo H. These compositions may further comprise suitablecarriers, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art.

Isolated antibodies and polynucleotides are also provided. In certainembodiments, the isolated antibodies and polynucleotides aresubstantially pure.

In one embodiment, anti-SSEA-3/SSEA-4/Globo H antibodies are monoclonal.In another embodiment, fragments of the anti-SSEA-3/SSEA-4/Globo Hantibodies (e.g., Fab, Fab′-SH and F(ab′)2 fragments) are provided.These antibody fragments can be created by traditional means, such asenzymatic digestion, or may be generated by recombinant techniques. Suchantibody fragments may be chimeric, humanized, or human. These fragmentsare useful for the diagnostic and therapeutic purposes set forth below.

A variety of methods are known in the art for generating phage displaylibraries from which an antibody of interest can be obtained. One methodof generating antibodies of interest is through the use of a phageantibody library as described in Lee et al., J. Mol. Biol. (2004),340(5): 1073-93.

The anti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be madeby using combinatorial libraries to screen for synthetic antibody cloneswith the desired activity or activities. In principle, syntheticantibody clones are selected by screening phage libraries containingphage that display various fragments of antibody variable region (Fv)fused to phage coat protein. Such phage libraries are panned by affinitychromatography against the desired antigen. Clones expressing Fvfragments capable of binding to the desired antigen are adsorbed to theantigen and thus separated from the non-binding clones in the library.The binding clones are then eluted from the antigen, and can be furtherenriched by additional cycles of antigen adsorption/elution. Any of theanti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be obtainedby designing a suitable antigen screening procedure to select for thephage clone of interest followed by construction of a full lengthanti-SSEA-3/SSEA-4/Globo H antibody clone using the Fv sequences fromthe phage clone of interest and suitable constant region (Fc) sequencesdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-SSEA-3/SSEA-4/Globo H clones is desired, the subject isimmunized with SSEA-3/SSEA-4/Globo H to generate an antibody response,and spleen cells and/or circulating B cells or other peripheral bloodlymphocytes (PBLs) are recovered for library construction. In oneembodiment, a human antibody gene fragment library biased in favor ofanti-human SSEA-3/SSEA-4/Globo H clones is obtained by generating ananti-human SSEA-3/SSEA-4/Globo H antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such thatSSEA-3/SSEA-4/Globo H immunization gives rise to B cells producing humanantibodies against SSEA-3/SSEA-4/Globo H. The generation of humanantibody-producing transgenic mice is described below.

Additional enrichment for anti-SSEA-3/SSEA-4/Globo H reactive cellpopulations can be obtained by using a suitable screening procedure toisolate B cells expressing SSEA-3/SSEA-4/Globo H-specific antibody,e.g., by cell separation with SSEA-3/SSEA-4/Globo H affinitychromatography or adsorption of cells to fluorochrome-labeledSSEA-3/SSEA-4/Globo H followed by flow-activated cell sorting (FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in whichSSEA-3/SSEA-4/Globo H is not antigenic. For libraries incorporating invitro antibody gene construction, stem cells are harvested from thesubject to provide nucleic acids encoding unrearranged antibody genesegments. The immune cells of interest can be obtained from a variety ofanimal species, such as human, mouse, rat, lagomorpha, luprine, canine,feline, porcine, bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, the library diversity is maximized byusing PCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 1012 clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (Kd-1 of about 10-8 M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

Screening of the libraries can be accomplished by any art-knowntechnique. For example, SSEA-3/SSEA-4/Globo H targets can be used tocoat the wells of adsorption plates, expressed on host cells affixed toadsorption plates or used in cell sorting, or conjugated to biotin forcapture with streptavidin-coated beads, or used in any other art-knownmethod for panning phage display libraries.

The phage library samples are contacted with immobilizedSSEA-3/SSEA-4/Globo H under conditions suitable for binding of at leasta portion of the phage particles with the adsorbent. Normally, theconditions, including pH, ionic strength, temperature and the like areselected to mimic physiological conditions. The phages bound to thesolid phase are washed and then eluted by acid, e.g. as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or byalkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597(1991), or by SSEA-3/SSEA-4/Globo H antigen competition, e.g. in aprocedure similar to the antigen competition method of Clackson et al.,Nature, 352: 624-628 (1991). Phages can be enriched from about 20× toabout 1,000-fold in a single round of selection. Moreover, the enrichedphages can be grown in bacterial culture and subjected to further roundsof selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, forSSEA-3/SSEA-4/Globo H. However, random mutation of a selected antibody(e.g. as performed in some of the affinity maturation techniquesdescribed above) is likely to give rise to many mutants, most binding toantigen, and a few with higher affinity. With limitingSSEA-3/SSEA-4/Globo H, rare high affinity phage could be competed out.To retain all the higher affinity mutants, phages can be incubated withexcess biotinylated SSEA-3/SSEA-4/Globo H, but with the biotinylatedSSEA-3/SSEA-4/Globo H at a concentration of lower molarity than thetarget molar affinity constant for SSEA-3/SSEA-4/Globo H. The highaffinity-binding phages can then be captured by streptavidin-coatedparamagnetic beads. Such “equilibrium capture” allows the antibodies tobe selected according to their affinities of binding, with sensitivitythat permits isolation of mutant clones with as little as two-foldhigher affinity from a great excess of phages with lower affinity.Conditions used in washing phages bound to a solid phase can also bemanipulated to discriminate on the basis of dissociation kinetics.

Anti-SSEA-3/SSEA-4/Globo H clones may be activity selected. In oneembodiment, the invention provides anti-SSEA-3/SSEA-4/Globo H antibodiesthat block the binding between a SSEA-3/SSEA-4/Globo H ligand andSSEA-3/SSEA-4/Globo H, but do not block the binding between aSSEA-3/SSEA-4/Globo H ligand and a second protein. Fv clonescorresponding to such anti-SSEA-3/SSEA-4/Globo H antibodies can beselected by (1) isolating anti-SSEA-3/SSEA-4/Globo H clones from a phagelibrary as described in Section B(I)(2) above, and optionally amplifyingthe isolated population of phage clones by growing up the population ina suitable bacterial host; (2) selecting SSEA-3/SSEA-4/Globo H and asecond protein against which blocking and non-blocking activity,respectively, is desired; (3) adsorbing the anti-SSEA-3/SSEA-4/Globo Hphage clones to immobilized SSEA-3/SSEA-4/Globo H; (4) using an excessof the second protein to elute any undesired clones that recognizeSSEA-3/SSEA-4/Globo H-binding determinants which overlap or are sharedwith the binding determinants of the second protein; and (5) eluting theclones which remain adsorbed following step (4). Optionally, clones withthe desired blocking/non-blocking properties can be further enriched byrepeating the selection procedures described herein one or more times.

DNA encoding the Fv clones of the invention is readily isolated andsequenced using conventional procedures (e.g. by using oligonucleotideprimers designed to specifically amplify the heavy and light chaincoding regions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In one embodiment, a Fvclone derived from human variable DNA is fused to human constant regionDNA to form coding sequence(s) for all human, full or partial lengthheavy and/or light chains.

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (Kd-1 of about 106 to 107 M−1), but affinitymaturation can also be mimicked in vitro by constructing and reselectingfrom secondary libraries as described in Winter et al. (1994), supra.For example, mutation can be introduced at random in vitro by usingerror-prone polymerase (reported in Leung et al., Technique, 1: 11-15(1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896(1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). Additionally, affinity maturation can be performed byrandomly mutating one or more CDRs, e.g. using PCR with primers carryingrandom sequence spanning the CDR of interest, in selected individual Fvclones and screening for higher affinity clones. WO 9607754 (published14 Mar. 1996) described a method for inducing mutagenesis in acomplementarity determining region of an immunoglobulin light chain tocreate a library of light chain genes. Another effective approach is torecombine the VH or VL domains selected by phage display withrepertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10-9 M range.

Other Methods of Generating Anti-SSEA-3/SSEA-4/Globo H Antibodies

Other methods of generating and assessing the affinity of antibodies arewell known in the art and are described, e.g., in Kohler et al., Nature256: 495 (1975); U.S. Pat. No. 4,816,567; Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986; Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987; Munson et al., Anal. Biochem., 107:220 (1980); Engels etal., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989); Abrahmsen et al.,EMBO J., 4: 3901 (1985); Methods in Enzymology, vol. 44 (1976); Morrisonet al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).

General Methods

Accordingly, one aspect of the present disclosure features an isolatedantibody triple-targeting Globo H, SSEA3 and SSEA-4. Thetriple-targeting antibody specifically binds toFuca1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (Globo H hexasaccharide) andGalβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-3 pentasaccharide) andNeu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide).In one example, the triple-targeting antibody is mAb 651.

Another aspect of the present disclosure features an isolated antibodydual-targeting Globo H and SSEA3. The dual-targeting antibodyspecifically binds to Fuca1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (GloboH hexasaccharide) and Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-3pentasaccharide), In one example, the dual-targeting antibody is mAb273.

In yet another aspect, the present disclosure features an isolatedantibody specific to SSEA-4. The anti-SSEA-4 antibody binds toNeu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4 hexasaccharide).In some examples, the antibody is capable of bindingNeu5Gca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (an analogue of SSEA-4hexasaccharide). Preferably, the antibody is not a mouse IgG3(e.g., mAbMC-831-70), and the antibody is not a mouse IgM (e.g., anti-RM1).Examples of the antibodies include, but are not limited to, mAbs 45 and48.

Another aspect of the present disclosure features an isolated antibodyspecific to SSEA-4 and fragments thereof. The anti-SSEA-4 antibody bindsto Neu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (SSEA-4hexasaccharide) and Neu5Aca2→3Galβ1→3GalNAcβ1→3Galα1(fragment of SSEA-4hexasaccharide). In some examples, the antibody is capable ofNeu5Aca2→3Galβ1→3GalNAcβ1→3Galβ1. In some examples, the antibody iscapable of Neu5Gca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1 (an analogueof SSEA-4 hexasaccharide). In one example, the antibody is mAb 46.

Antibodies triple-targeting Globo H, SSEA-3 and SSEA-4, antibodiesdual-targeting Globo H and SSEA-3, and anti-SSEA-4 antibodies weredeveloped and disclosed herein. The antibodies according to thedisclosure can be used in therapeutics, diagnosis or as a research tool.

Accordingly, one aspect of the present disclosure relates to acomposition of a homogeneous population of monoclonal antibodiescomprising a single, uniform N-glycan on Fc, wherein the structure is anoptimized N-glycan structure for enhancing the efficacy of effector cellfunction.

In preferred embodiments, the N-glycan is attached to the Asn-297 of theFc region.

In preferred embodiments, wherein the N-glycan consists of the structureof Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂.

The glycoantibodies described herein may be produced in vitro. Theglycoantibodies may be generated by Fc glycoengineering. In certainembodiments, the glycoantibodies are enzymatically or chemoenzymaticallyengineered from the monoclonal antibodies obtained by mammalian cellculturing.

In some embodiments, the Fc region of the glycoantibodies describedherein exhibits an increased binding affinity for FcγRIIA or FcγRIIIArelative to a wild-type Fc region in the corresponding monoclonalantibodies.

In some embodiments, the glycoantibodies described herein exhibit anenhanced antibody-dependent cell mediated cytotoxicity (ADCC) activityrelative to wild-type immunoglobulins.

In some embodiments, the glycoantibodies are selected from a groupconsisting of human IgG1, IgG2, IgG3, and IgG4. The monoclonalantibodies may be humanized, human or chimeric.

The glycoantibodies described herein may bind to an antigen associatedwith cancers, autoimmune disorders, inflammatory disorders or infectiousdiseases. Exemplary cancer associated antigens can include, for example,Globo-H, SSEA-3, SSEA-4.

In other aspects, the antibodies disclosed herein can detect glycanvariants and derivatives. For example, the reducing end of the glycan isfree or linked to a tail which is natural (e.g. SSEA4 glycolipid) ornon-natural (e.g. a linker for making glycan array or for conjugationfor diagnostic purposes). All these derivatives can be recognized by theantibody.

In certain diagnostic and array embodiments, the antibodies of thisinvention can therefore detect not only the glycan described herein, butalso oxidized variants thereof. The antibodies of this invention canalso detect conjugation products to said oxidized variants.

In certain aspects, the disclosure provides isolated humanizedmonoclonal glycoantibody that specifically binds toNeu5Aca2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, and oxidized variantsthereof, and conjugation products to said oxidized variants, andoxidized variants thereof, and conjugation products to said oxidizedvariants; wherein said oxidized variants are the conversion products ofthe glycan primary alcohols to carbonyls, and wherein the conjugationproducts are the conversion products of carbonyls to imines with aprimary or secondary amine moiety.

For example, the glycans comprising primary alcohols can be converted toan oxidized variant by methods known to those skilled in the art. As anon-limiting example, a primary alcohol on a galactose can be convertedto an aldehyde by contacting the glycan with an oxidant, e.g. sodiumperiodate (sodium m-periodate), or another salt of periodate (e.g.,potassium, ammonium, manganese, lithium). One or a plurality of sugarmoieties in the glycan can be oxidized. The concentration of oxidant canbe 1 micromolar, 5 micromolar, 10 micromolar, 25 micromolar, 50micromolar, 100 micromolar, 200 micromolar, 500 micromolar, 750micromolar, 1 millimolar, 5 millimolar, 10 millimolar, 25 millimolar, 50millimolar, 100 millimolar, or 500 millimolar in water or a suitablebuffer. The temperature can be from 5 to 45 degrees Celsius, preferably15 to 40 degrees Celsius, more preferably 35 to 40 degrees Celsius. Thereaction time can be from 10 seconds to 20 minutes, preferably from 30seconds to 10 minutes. Suitable buffers can include or exclude saline,phosphate, CHES, MES, borate, acetate, carbonate, formate, citrate,oxalate. Preferably, mildly acidic buffers are used. Preferably, bufferswithout TRIS or glycine or free sugars are used as these will compete inthe reaction. The conversion can be purified by dialysis or centrifugaldialysis by methods known those skilled in the art.

The conjugation products can be formed from the reaction of the oxidizedproducts with an appropriate amine, hydrazine, hydrazide, or oxo-amineby methods known to those skilled in the art, and as described in G.Hermanson, Bioconjugate Techniques, 3^(rd) Ed., ISBN: 978-0-12-382239-0,Academic Press, 2013, herein incorporated by reference. As anon-limiting example, a primary amine can be reacted to a glycan with asingle aldehyde functional group formed from the periodate-oxidizedprimary alcohol of a galactose within the glycan. The net product wouldbe an imine. The imine can be optionally further reduced to an alcoholby methods known the those skilled in the art, e.g. cyanoborohydridereduction, to form a more stable conjugation product to hydrolysis. Insome aspects, the amine, hydrazine, hydrazide, or oxo-amine can befurther covalently linked to an array, a reporter molecule, or a biotinfor further modification of the conjugation product. In some aspects,the reporter molecule can be a fluorescent molecule. In some aspects,the reporter molecule can be a radiolabelled molecule. In some aspects,the reporter molecule can be a molecule with a unique spectralcharacteristic (e.g., IR spectra, Raman spectra, or NMR spectra). Insome aspects, the array can be a solid surface, a chemically modifiedsurface, a polymer-coated surface, a bead, a gel, a particle, or ananoparticle. In some aspects, the nanoparticle can be fluorescent orexhibit photoluminescence. In some aspects, the conjugation products canbe the conversion products of carbonyls to imines with a primary orsecondary amine moiety.

In general, the invention provides affinity-matured SSEA-3/SSEA-4/GloboH antibodies. These antibodies have increased affinity and specificityfor SSEA-3/SSEA-4/Globo H. This increase in affinity and sensitivitypermits the molecules of the invention to be used for applications andmethods that are benefited by (a) the increased sensitivity of themolecules of the invention and/or (b) the tight binding ofSSEA-3/SSEA-4/Globo H by the molecules of the invention.

In one aspect, SSEA4/SSEA3/GloboH are three glycans that arespecifically expressed for cancer cells and cancer stem cells. Knockdownof beta-3-GalT5, the key enzyme for the synthesis of these threeglycolipids, causes apoptosis of cancer cells, but not normal cells.Antibodies, especially glycoantibodies against SSEA4 preferentially orspecifically and/or against SSEA3/SSEA4/GloboH simultaneously areeffective cancer therapeutic agents. In another aspect, the threeglycans, SSEA4/SSEA3/GloboH, especially SSEA3, are useful as cancer stemcell markers.

In one aspect, SSEA4 and/or SSEA4/SSEA3/GloboH in combination are usefulas therapeutic targets for the treatment of different cancers, includingfor example, brain cancer, lung cancer, breast cancer, oral cancer,esophageal cancer, stomach cancer, liver cancer, bile duct cancer,pancreatic cancer, colon cancer, kidney cancer, bone cancer(osteosarcoma), skin cancer, cervical cancer, ovarian cancer, andprostate cancer.

In one embodiment, human or humanized therapeutic antibodies againstSSEA4 expressed on the cell surface of these exemplary cancer types areprovided.

In another embodiment, human or humanized therapeutic antibodies againstSSEA3/SSEA4/Globo-H simultaneously expressed on the cell surface ofthese exemplary cancer types are provided.

Additionally, the present disclosure is also directed to immunogenicconjugate compositions targeting the SSEA-3/SSEA-4/Globo H associatedepitopes (natural and modified) which can elicit antibodies and/orbinding fragment production useful for modulating the globoseriesglycosphingolipid synthesis. Moreover, the present disclosure is alsodirected to the method of using the compositions described herein forthe treatment or detection of hyperproliferative diseases and/orconditions.

In one embodiment, SSEA-3/SSEA-4/Globo H antibodies that are useful fortreatment of SSEA-3/SSEA-4/Globo H-mediated disorders in which a partialor total blockade of one or more SSEA-3/SSEA-4/Globo H activities isdesired. In one embodiment, the anti SSEA-3/SSEA-4/Globo H antibodies ofthe invention are used to treat cancer.

The anti-SSEA-3/SSEA-4/Globo H antibodies of the invention permit thesensitive and specific detection of the epitopes in immunoassays such assandwich assays, immunoprecipitations, ELISAs, or immunomicroscopywithout the need for mass spectrometry or genetic manipulation. In turn,this provides a significant advantage in both observing and elucidatingthe normal functioning of these pathways and in detecting when thepathways are functioning aberrantly.

The SSEA-3/SSEA-4/Globo H antibodies of the invention can also be usedto determine the role in the development and pathogenesis of disease.For example, as described above, the SSEA-3/SSEA-4/Globo H antibodies ofthe invention can be used to determine whether the TACAs are normallytemporally expressed which can be correlated with one or more diseasestates.

The SSEA-3/SSEA-4/Globo H antibodies of the invention can further beused to treat diseases in which one or more SSEA-3/SSEA-4/Globo Hs areaberrantly regulated or aberrantly functioning without interfering withthe normal activity of SSEA-3/SSEA-4/Globo Hs for which theanti-SSEA-3/SSEA-4/Globo H antibodies of the invention are not specific.

In another aspect, the anti-SSEA-3/SSEA-4/Globo H antibodies of theinvention find utility as reagents for detection of cancer states invarious cell types and tissues.

In yet another aspect, the present anti-SSEA-3/SSEA-4/Globo H antibodiesare useful for the development of SSEA-3/SSEA-4/Globo H antagonists withblocking activity patterns similar to those of the subject antibodies ofthe invention. For example, anti-SSEA-3/SSEA-4/Globo H antibodies of theinvention can be used to determine and identify other antibodies thathave the same SSEA-3/SSEA-4/Globo H binding characteristics and/orcapabilities of blocking SSEA-3/SSEA-4/Globo H-pathways.

As a further example, anti-SSEA-3/SSEA-4/Globo H antibodies of theinvention can be used to identify other anti-SSEA-3/SSEA-4/Globo Hantibodies that bind substantially the same antigenic determinant(s) ofSSEA-3/SSEA-4/Globo H as the antibodies exemplified herein, includinglinear and conformational epitopes.

The anti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be usedin assays based on the physiological pathways in whichSSEA-3/SSEA-4/Globo H is involved to screen for small moleculeantagonists of SSEA-3/SSEA-4/Globo H which will exhibit similarpharmacological effects in blocking the binding of one or more bindingpartners to SSEA-3/SSEA-4/Globo H as the antibody does.

Generation of antibodies can be achieved using routine skills in theart, including those described herein, such as the hybridoma techniqueand screening of phage displayed libraries of binder molecules. Thesemethods are well-established in the art.

Briefly, the anti-SSEA-3/SSEA-4/Globo H antibodies of the invention canbe made by using combinatorial libraries to screen for syntheticantibody clones with the desired activity or activities. In principle,synthetic antibody clones are selected by screening phage librariescontaining phage that display various fragments of antibody variableregion (Fv) fused to phage coat protein. Such phage libraries are pannedby affinity chromatography against the desired antigen. Clonesexpressing Fv fragments capable of binding to the desired antigen areadsorbed to the antigen and thus separated from the non-binding clonesin the library. The binding clones are then eluted from the antigen, andcan be further enriched by additional cycles of antigenadsorption/elution. Any of the anti-SSEA-3/SSEA-4/Globo H antibodies ofthe invention can be obtained by designing a suitable antigen screeningprocedure to select for the phage clone of interest followed byconstruction of a full length anti-SSEA-3/SSEA-4/Globo H antibody cloneusing the Fv sequences from the phage clone of interest and suitableconstant region (Fc) sequences described in Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3.

In one embodiment, anti-SSEA-3/SSEA-4/Globo H antibodies of theinvention are monoclonal. Also encompassed within the scope of theinvention are antibody fragments such as Fab, Fab′, Fab′-SH and F(ab′)2fragments, and variations thereof, of the anti-SSEA-3/SSEA-4/Globo Hantibodies provided herein. These antibody fragments can be created bytraditional means, such as enzymatic digestion, or may be generated byrecombinant techniques. Such antibody fragments may be chimeric, humanor humanized. These fragments are useful for the experimental,diagnostic, and therapeutic purposes set forth herein.

Monoclonal antibodies can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-SSEA-3/SSEA-4/Globo H monoclonal antibodies of the inventioncan be made using a variety of methods known in the art, including thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or alternatively they may be made by recombinant DNA methods(e.g., U.S. Pat. No. 4,816,567).

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Host cells include,but are not limited to, cells of either prokaryotic or eukaryotic(generally mammalian) origin. It will be appreciated that constantregions of any isotype can be used for this purpose, including IgG, IgM,IgA, IgD, and IgE constant regions, and that such constant regions canbe obtained from any human or animal species.

Generating Antibodies Using Prokaryotic Host Cells

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λ GEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 Δ fhuA (Δ tonA) ptr3 lac Iq lacL8 Δ ompT Δ (nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776(ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, growth occurs at a temperature rangeincluding, but not limited to, about 20° C. to about 39° C., about 25°C. to about 37° C., and at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH can be from about 6.8 to about 7.4, orabout 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In one embodiment, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, for example about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (a common carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected generally is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II (e.g., primate metallothionein genes), adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene may first beidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR.Appropriate host cells when wild-type DHFR is employed include, forexample, the Chinese hamster ovary (CHO) cell line deficient in DHFRactivity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. Virtually all eukaryotic genes havean AT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3 end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellscan be controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding an antibody polypeptide of the inventionby higher eukaryotes can often be increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is generally located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are generally removed, forexample, by centrifugation or ultrafiltration. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a generally acceptable purification technique. The suitability ofaffinity reagents such as protein A as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is present inthe antibody. Protein A can be used to purify antibodies that are basedon human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification steps, as necessary, for example by low pH hydrophobicinteraction chromatography using an elution buffer at a pH between about2.5-4.5, generally performed at low salt concentrations (e.g., fromabout 0-0.25M salt).

It should be noted that, in general, techniques and methodologies forpreparing antibodies for use in research, testing and clinical use arewell-established in the art, consistent with the above and/or as deemedappropriate by one skilled in the art for the particular antibody ofinterest.

Activity Assays

Antibodies of the invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

Purified antibodies can be further characterized by a series of assaysincluding, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

Where necessary, antibodies are analyzed for their biological activity.In some embodiments, antibodies of the invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays,chemiluminescent immunoassays, nanoparticle immunoassays, aptamerimmunoassays, and protein A immunoassays.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)2 fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab′)2 fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)2 fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human Antibodies

Human anti-SSEA-3/SSEA-4/Globo H antibodies of the invention can beconstructed by combining Fv clone variable domain sequence(s) selectedfrom human-derived phage display libraries with known human constantdomain sequences(s) as described above. Alternatively, human monoclonalanti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be made bythe hybridoma method. Human myeloma and mouse-human heteromyeloma celllines for the production of human monoclonal antibodies have beendescribed, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is forSSEA-3/SSEA-4/Globo H including a specific lysine linkage and the otheris for any other antigen. In certain embodiments, bispecific antibodiesmay bind to two different SSEA-3/SSEA-4/Globo Hs having two differentlysine linkages. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments (e.g. F(ab′)2 bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different embodiment, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the CH3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The dimerization domain comprises (or consists of), forexample, an Fc region or a hinge region. In this scenario, the antibodywill comprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. In one embodiment, a multivalentantibody comprises (or consists of), for example, three to about eight,or four antigen binding sites. The multivalent antibody comprises atleast one polypeptide chain (for example, two polypeptide chains),wherein the polypeptide chain(s) comprise two or more variable domains.For instance, the polypeptide chain(s) may compriseVD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is asecond variable domain, Fc is one polypeptide chain of an Fc region, X1and X2 represent an amino acid or polypeptide, and n is 0 or 1. Forinstance, the polypeptide chain(s) may comprise: VH-CH1-flexiblelinker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. Themultivalent antibody herein may further comprise at least two (forexample, four) light chain variable domain polypeptides. The multivalentantibody herein may, for instance, comprise from about two to abouteight light chain variable domain polypeptides. The light chain variabledomain polypeptides contemplated here comprise a light chain variabledomain and, optionally, further comprise a CL domain. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics. The amino acidalterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table A under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table A,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE A Original Exemplary Preferred Residue Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val;Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

-   -   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe        (F), Trp (W), Met (M)    -   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr        (Y), Asn (N), Gln (O)    -   (3) acidic: Asp (D), Glu (E)    -   (4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions toat least part of a phage coat protein (e.g., the gene III product ofM13) packaged within each particle. The phage-displayed variants arethen screened for their biological activity (e.g. binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, scanning mutagenesis (e.g., alanine scanning)can be performed to identify hypervariable region residues contributingsignificantly to antigen binding. Alternatively, or additionally, it maybe beneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and antigen.Such contact residues and neighboring residues are candidates forsubstitution according to techniques known in the art, including thoseelaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening using techniques known in the art,including those described herein, and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

Immunoconjugates

In another aspect, the invention provides immunoconjugates, orantibody-drug conjugates (ADC), comprising an antibody conjugated to acytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubutin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

Antibody Derivatives

Antibodies of the invention can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. In one embodiment, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, the polymers can be the sameor different molecules. In general, the number and/or type of polymersused for derivatization can be determined based on considerationsincluding, but not limited to, the particular properties or functions ofthe antibody to be improved, whether the antibody derivative will beused in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, including, butnot limited to those with complementary activities that do not adverselyaffect each other. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the invention may be used in, for example, in vitro, exvivo and in vivo therapeutic methods. Antibodies of the invention can beused as an antagonist to partially or fully block the specific antigenactivity in vitro, ex vivo and/or in vivo. Moreover, at least some ofthe antibodies of the invention can neutralize antigen activity fromother species. Accordingly, antibodies of the invention can be used toinhibit a specific antigen activity, e.g., in a cell culture containingthe antigen, in human subjects or in other mammalian subjects having theantigen with which an antibody of the invention cross-reacts (e.g.chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). Inone embodiment, an antibody of the invention can be used for inhibitingantigen activities by contacting the antibody with the antigen such thatantigen activity is inhibited. In one embodiment, the antigen is a humanprotein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor inhibiting an antigen in a subject suffering from a disorder inwhich the antigen activity is detrimental, comprising administering tothe subject an antibody of the invention such that the antigen activityin the subject is inhibited. In one embodiment, the antigen is a humanprotein molecule and the subject is a human subject. Alternatively, thesubject can be a mammal expressing the antigen with which an antibody ofthe invention binds. Still further the subject can be a mammal intowhich the antigen has been introduced (e.g., by administration of theantigen or by expression of an antigen transgene). An antibody of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, an antibody of the invention can be administered toa non-human mammal expressing an antigen with which the antibodycross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Antibodies of the invention can be used to treat,inhibit, delay progression of, prevent/delay recurrence of, ameliorate,or prevent diseases, disorders or conditions associated with abnormalexpression and/or activity of SSEA-3/SSEA-4/Globo Hs andSSEA-3/SSEA-4/Globo Hated proteins, including but not limited to cancer,muscular disorders, ubiquitin-pathway-related genetic disorders,immune/inflammatory disorders, neurological disorders, and otherubiquitin pathway-related disorders.

In one aspect, a blocking antibody of the invention is specific for aSSEA-3/SSEA-4/Globo H.

In certain embodiments, an immunoconjugate comprising an antibody of theinvention conjugated with a cytotoxic agent is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by cells expressing one or moreproteins on their cell surface which are associated withSSEA-3/SSEA-4/Globo H, resulting in increased therapeutic efficacy ofthe immunoconjugate in killing the target cell with which it isassociated. In one embodiment, the cytotoxic agent targets or interfereswith nucleic acid in the target cell. Examples of such cytotoxic agentsinclude any of the chemotherapeutic agents noted herein (such as amaytansinoid or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, and/oradjuvant/therapeutic agents (e.g., steroids). For instance, an antibodyof the invention may be combined with an anti-inflammatory and/orantiseptic in a treatment scheme, e.g. in treating any of the diseasesdescribed herein, including cancer, muscular disorders,ubiquitin-pathway-related genetic disorders, immune/inflammatorydisorders, neurological disorders, and other ubiquitin pathway-relateddisorders. Such combined therapies noted above include combinedadministration (where the two or more agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,and/or following, administration of the adjunct therapy or therapies.

An antibody of the invention (and adjunct therapeutic agent) can beadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, e.g. by injections, suchas intravenous or subcutaneous injections, depending in part on whetherthe administration is brief or chronic.

The location of the binding target of an antibody of the invention maybe taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described in Marasco, Gene Therapy 4: 11-15(1997); Kontermann, Methods 34: 163-170 (2004); U.S. Pat. Nos. 6,004,940and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, andPCT Publication No. WO2003/077945. Intracellular expression of anintrabody is effected by introducing a nucleic acid encoding the desiredantibody or antigen-binding portion thereof (lacking the wild-typeleader sequence and secretory signals normally associated with the geneencoding that antibody or antigen-binding fragment) into a target cell.Any standard method of introducing nucleic acids into a cell may beused, including, but not limited to, microinjection, ballisticinjection, electroporation, calcium phosphate precipitation, liposomes,and transfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest. One or morenucleic acids encoding all or a portion of an anti-SSEA-3/SSEA-4/Globo Hantibody of the invention can be delivered to a target cell, such thatone or more intrabodies are expressed which are capable of intracellularbinding to a SSEA-3/SSEA-4/Globo H and modulation of one or moreSSEA-3/SSEA-4/Globo H-mediated cellular pathways.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the binding domain of the target protein is generallyadvantageous. For example, based upon the variable-region sequences ofan antibody, peptide molecules can be designed that retain the abilityto bind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

Entry of modulator polypeptides into target cells can be enhanced bymethods known in the art. For example, certain sequences, such as thosederived from HIV Tat or the Antennapedia homeodomain protein are able todirect efficient uptake of heterologous proteins across cell membranes.See, e.g., Chen et al., Proc. Natl. Acad. Sci. USA (1999), 96:4325-4329.

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or antigen-binding fragmentthereof to traverse the blood-brain barrier. Certain neurodegenerativediseases are associated with an increase in permeability of theblood-brain barrier, such that the antibody or antigen-binding fragmentcan be readily introduced to the brain. When the blood-brain barrierremains intact, several art-known approaches exist for transportingmolecules across it, including, but not limited to, physical methods,lipid-based methods, and receptor and channel-based methods.

Physical methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9: 398-406 (2002)), interstitialinfusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc.Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a deliverydevice in the brain (see, e.g., Gill et al., Nature Med. 9: 589-595(2003); and Gliadel Wafers™, Guildford Pharmaceutical). Methods ofcreating openings in the barrier include, but are not limited to,ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086),osmotic pressure (e.g., by administration of hypertonic mannitol(Neuwelt, E. A., Implication of the Blood-Brain Barrier and itsManipulation, Vols 1 & 2, Plenum Press, N.Y. (1989))), permeabilizationby, e.g., bradykinin or permeabilizer A-7 (see, e.g., U.S. Pat. Nos.5,112,596, 5,268,164, 5,506,206, and 5,686,416), and transfection ofneurons that straddle the blood-brain barrier with vectors containinggenes encoding the antibody or antigen-binding fragment (see, e.g., U.S.Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,encapsulating the antibody or antigen-binding fragment in liposomes thatare coupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody orantigen-binding fragment in low-density lipoprotein particles (see,e.g., U.S. Patent Application Publication No. 20040204354) orapolipoprotein E (see, e.g., U.S. Patent Application Publication No.20040131692).

Receptor and channel-based methods of transporting the antibody orantigen-binding fragment across the blood-brain barrier include, but arenot limited to, using glucocorticoid blockers to increase permeabilityof the blood-brain barrier (see, e.g., U.S. Patent ApplicationPublication Nos. 2002/0065259, 2003/0162695, and 2005/0124533);activating potassium channels (see, e.g., U.S. Patent ApplicationPublication No. 2005/0089473), inhibiting ABC drug transporters (see,e.g., U.S. Patent Application Publication No. 2003/0073713); coatingantibodies with a transferrin and modulating activity of the one or moretransferrin receptors (see, e.g., U.S. Patent Application PublicationNo. 2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat.No. 5,004,697).

The antibody composition of the invention would be formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The antibody need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of antibodies of the invention present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents such as chemotherapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition effectivefor treating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice. Moreover,the article of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises anantibody of the invention; and (b) a second container with a compositioncontained therein, wherein the composition comprises a further cytotoxicor otherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Pharmaceutical Compositions and Formulations

After preparation of the antibodies as described herein,“pre-lyophilized formulation” can be produced. The antibody forpreparing the formulation is preferably essentially pure and desirablyessentially homogeneous (i.e. free from contaminating proteins etc).“Essentially pure” protein means a composition comprising at least about90% by weight of the protein, based on total weight of the composition,preferably at least about 95% by weight. “Essentially homogeneous”protein means a composition comprising at least about 99% by weight ofprotein, based on total weight of the composition. In certainembodiments, the protein is an antibody.

The amount of antibody in the pre-lyophilized formulation is determinedtaking into account the desired dose volumes, mode(s) of administrationetc. Where the protein of choice is an intact antibody (a full-lengthantibody), from about 2 mg/mL to about 50 mg/mL, preferably from about 5mg/mL to about 40 mg/mL and most preferably from about 20-30 mg/mL is anexemplary starting protein concentration. The protein is generallypresent in solution. For example, the protein may be present in apH-buffered solution at a pH from about 4-8, and preferably from about5-7. Exemplary buffers include histidine, phosphate, Tris, citrate,succinate and other organic acids. The buffer concentration can be fromabout 1 mM to about 20 mM, or from about 3 mM to about 15 mM, depending,for example, on the buffer and the desired isotonicity of theformulation (e.g. of the reconstituted formulation). The preferredbuffer is histidine in that, as demonstrated below, this can havelyoprotective properties. Succinate was shown to be another usefulbuffer.

The lyoprotectant is added to the pre-lyophilized formulation. Inpreferred embodiments, the lyoprotectant is a non-reducing sugar such assucrose or trehalose. The amount of lyoprotectant in the pre-lyophilizedformulation is generally such that, upon reconstitution, the resultingformulation will be isotonic. However, hypertonic reconstitutedformulations may also be suitable. In addition, the amount oflyoprotectant must not be too low such that an unacceptable amount ofdegradation/aggregation of the protein occurs upon lyophilization. Wherethe lyoprotectant is a sugar (such as sucrose or trehalose) and theprotein is an antibody, exemplary lyoprotectant concentrations in thepre-lyophilized formulation are from about 10 mM to about 400 mM, andpreferably from about 30 mM to about 300 mM, and most preferably fromabout 50 mM to about 100 mM.

The ratio of protein to lyoprotectant is selected for each protein andlyoprotectant combination. In the case of an antibody as the protein ofchoice and a sugar (e.g., sucrose or trehalose) as the lyoprotectant forgenerating an isotonic reconstituted formulation with a high proteinconcentration, the molar ratio of lyoprotectant to antibody may be fromabout 100 to about 1500 moles lyoprotectant to 1 mole antibody, andpreferably from about 200 to about 1000 moles of lyoprotectant to 1 moleantibody, for example from about 200 to about 600 moles of lyoprotectantto 1 mole antibody.

In preferred embodiments of the invention, it has been found to bedesirable to add a surfactant to the pre-lyophilized formulation.Alternatively, or in addition, the surfactant may be added to thelyophilized formulation and/or the reconstituted formulation. Exemplarysurfactants include nonionic surfactants such as polysorbates (e.g.polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton; sodiumdodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside;lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, orcetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palnidopropyl-, or isostearamidopropyl-betaine (e.glauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). The amount ofsurfactant added is such that it reduces aggregation of thereconstituted protein and minimizes the formation of particulates afterreconstitution. For example, the surfactant may be present in thepre-lyophilized formulation in an amount from about 0.001-0.5%, andpreferably from about 0.005-0.05%.

In certain embodiments of the invention, a mixture of the lyoprotectant(such as sucrose or trehalose) and a bulking agent (e.g. mannitol orglycine) is used in the preparation of the pre-lyophilizationformulation. The bulking agent may allow for the production of a uniformlyophilized cake without excessive pockets therein etc.

Other pharmaceutically acceptable carriers, excipients or stabilizerssuch as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the pre-lyophilizedformulation (and/or the lyophilized formulation and/or the reconstitutedformulation) provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; preservatives;co-solvents; antioxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

The pharmaceutical compositions and formulations described herein arepreferably stable. A “stable” formulation/composition is one in whichthe antibody therein essentially retains its physical and chemicalstability and integrity upon storage. Various analytical techniques formeasuring protein stability are available in the art and are reviewed inPeptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., MarcelDekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. DrugDelivery Rev. 10: 29-90 (1993). Stability can be measured at a selectedtemperature for a selected time period.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, lyophilization and reconstitution.Alternatively, sterility of the entire mixture may be accomplished byautoclaving the ingredients, except for protein, at about 120° C. forabout 30 minutes, for example.

After the protein, lyoprotectant and other optional components are mixedtogether, the formulation is lyophilized. Many different freeze-dryersare available for this purpose such as Hu1150® (Hull, USA) or GT20®(Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplishedby freezing the formulation and subsequently subliming ice from thefrozen content at a temperature suitable for primary drying. Under thiscondition, the product temperature is below the eutectic point or thecollapse temperature of the formulation. Typically, the shelftemperature for the primary drying will range from about −30 to 25° C.(provided the product remains frozen during primary drying) at asuitable pressure, ranging typically from about 50 to 250 mTorr. Theformulation, size and type of the container holding the sample (e.g.,glass vial) and the volume of liquid will mainly dictate the timerequired for drying, which can range from a few hours to several days(e.g. 40-60 hrs). A secondary drying stage may be carried out at about0-40° C., depending primarily on the type and size of container and thetype of protein employed. However, it was found herein that a secondarydrying step may not be necessary. For example, the shelf temperaturethroughout the entire water removal phase of lyophilization may be fromabout 15-30° C. (e.g., about 20° C.). The time and pressure required forsecondary drying will be that which produces a suitable lyophilizedcake, dependent, e.g., on the temperature and other parameters. Thesecondary drying time is dictated by the desired residual moisture levelin the product and typically takes at least about 5 hours (e.g. 10-15hours). The pressure may be the same as that employed during the primarydrying step. Freeze-drying conditions can be varied depending on theformulation and vial size.

In some instances, it may be desirable to lyophilize the proteinformulation in the container in which reconstitution of the protein isto be carried out in order to avoid a transfer step. The container inthis instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial. Asa general proposition, lyophilization will result in a lyophilizedformulation in which the moisture content thereof is less than about 5%,and preferably less than about 3%.

At the desired stage, typically when it is time to administer theprotein to the patient, the lyophilized formulation may be reconstitutedwith a diluent such that the protein concentration in the reconstitutedformulation is at least 50 mg/mL, for example from about 50 mg/mL toabout 400 mg/mL, more preferably from about 80 mg/mL to about 300 mg/mL,and most preferably from about 90 mg/mL to about 150 mg/mL. Such highprotein concentrations in the reconstituted formulation are consideredto be particularly useful where subcutaneous delivery of thereconstituted formulation is intended. However, for other routes ofadministration, such as intravenous administration, lower concentrationsof the protein in the reconstituted formulation may be desired (forexample from about 5-50 mg/mL, or from about 10-40 mg/mL protein in thereconstituted formulation). In certain embodiments, the proteinconcentration in the reconstituted formulation is significantly higherthan that in the pre-lyophilized formulation. For example, the proteinconcentration in the reconstituted formulation may be about 2-40 times,preferably 3-10 times and most preferably 3-6 times (e.g. at least threefold or at least four fold) that of the pre-lyophilized formulation.

Reconstitution generally takes place at a temperature of about 25° C. toensure complete hydration, although other temperatures may be employedas desired. The time required for reconstitution will depend, e.g., onthe type of diluent, amount of excipient(s) and protein. Exemplarydiluents include sterile water, bacteriostatic water for injection(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterilesaline solution, Ringer's solution or dextrose solution. The diluentoptionally contains a preservative. Exemplary preservatives have beendescribed above, with aromatic alcohols such as benzyl or phenol alcoholbeing the preferred preservatives. The amount of preservative employedis determined by assessing different preservative concentrations forcompatibility with the protein and preservative efficacy testing. Forexample, if the preservative is an aromatic alcohol (such as benzylalcohol), it can be present in an amount from about 0.1-2.0% andpreferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.Preferably, the reconstituted formulation has less than 6000 particlesper vial which are >10 μm size.

Therapeutic Applications

Described herein are therapeutic methods that include administering to asubject in need of such treatment a therapeutically effective amount ofa composition that includes one or more antibodies described herein.

In certain embodiments, the subject being treated is a mammal. Incertain embodiments, the subject is a human. In certain embodiments, thesubject is a domesticated animal, such as a dog, cat, cow, pig, horse,sheep, or goat. In certain embodiments, the subject is a companionanimal such as a dog or cat. In certain embodiments, the subject is alivestock animal such as a cow, pig, horse, sheep, or goat. In certainembodiments, the subject is a zoo animal. In another embodiment, thesubject is a research animal such as a rodent, dog, or non-humanprimate. In certain embodiments, the subject is a non-human transgenicanimal such as a transgenic mouse or transgenic pig.

In some embodiments, the subject (e.g., a human patient) in need of thetreatment is diagnosed with, suspected of having, or at risk for cancer.Examples of the cancer include, but are not limited to, brain cancer,lung cancer, breast cancer, oral cancer, esophagus cancer, stomachcancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer,kidney cancer, cervix cancer, ovary cancer and prostate cancer. In someembodiments, the cancer is brain cancer, lung cancer, breast cancer,ovarian cancer, prostate cancer, colon cancer, or pancreas cancer. Insome preferred embodiments, the cancer is brain cancer or glioblastomamultiforme (GBM) cancer.

In preferred embodiments, the antibody is capable of targeting Globo H,SSEA-3 and SSEA-4-expressing cancer cells. In some embodiments, theantibody is capable of targeting Globo H and SSEA on cancer cells. Insome embodiments, the antibody is capable of targeting SSEA in cancers.

Accordingly, the antibody is a triple-targeting antibody against GloboH, SSEA-3 and SSEA-4. In some embodiments, the antibodies are a mixtureof a dual-targeting antibody against Globo H and SSEA-3, and ananti-SSEA-4 antibody. In some embodiments, the antibodies are a mixtureof a triple-targeting antibody against Globo H, SSEA-3 and SSEA-4, andan anti-SSEA-4 antibody. In some embodiments, the antibody is a mixtureof an anti-Globo H, an anti-SSEA-3 and an anti-SSEA-4 antibody. In someembodiments, the antibody is a mixture of an anti-Globo H and ananti-SSEA-4 antibody. In some embodiments, the antibody is ananti-SSEA-4 antibody.

The treatment results in reduction of tumor size, elimination ofmalignant cells, prevention of metastasis, prevention of relapse,reduction or killing of disseminated cancer, prolongation of survivaland/or prolongation of time to tumor cancer progression.

In some embodiments, the treatment further comprises administering anadditional therapy to said subject prior to, during or subsequent tosaid administering of the antibodies. In some embodiments, theadditional therapy is treatment with a chemotherapeutic agent. In someembodiments, the additional therapy is radiation therapy.

The methods of the invention are particularly advantageous in treatingand preventing early stage tumors, thereby preventing progression to themore advanced stages resulting in a reduction in the morbidity andmortality associated with advanced cancer. The methods of the inventionare also advantageous in preventing the recurrence of a tumor or theregrowth of a tumor, for example, a dormant tumor that persists afterremoval of the primary tumor, or in reducing or preventing theoccurrence of a tumor.

In some embodiments, the methods as disclosed herein are useful for thetreatment or prevention of a cancer, for example where a cancer ischaracterized by increased Globo H, SSEA-3 and/or SSEA-4 expression. Insome embodiments the cancer comprises a cancer stem cell. In someembodiments, the cancer is a pre-cancer, and/or a malignant cancerand/or a therapy resistant cancer. In some embodiments, the cancer is abrain cancer.

For the methods of the invention, the cancer may be a solid tumor, e.g.,such as, breast cancer, colorectal cancer, rectal cancer, lung cancer,renal cell cancer, a glioma (e.g., anaplastic astrocytoma, anaplasticoligoastrocytoma, anaplastic oligodendroglioma, glioblastoma multiforme(GBM)), kidney cancer, prostate cancer, liver cancer, pancreatic cancer,soft-tissue sarcoma, carcinoid carcinoma, head and neck cancer,melanoma, and ovarian cancer. In one embodiment, the cancer is a braincancer or GBM. To practice the method disclosed herein, an effectiveamount of the pharmaceutical composition/formulation described above,containing at least one antibody described herein, can be administeredto a subject (e.g., a human) in need of the treatment via a suitableroute, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, inhalation or topical routes.Commercially available nebulizers for liquid formulations, including jetnebulizers and ultrasonic nebulizers are useful for administration.Liquid formulations can be directly nebulized and lyophilized powder canbe nebulized after reconstitution. Alternatively, the antibodies can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having cancer, whichinclude, but not limited to, brain cancer, lung cancer, breast cancer,oral cancer, esophagus cancer, stomach cancer, liver cancer, bile ductcancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer,ovary cancer and prostate cancer. A subject having cancer can beidentified by routine medical examination.

“An effective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size, gender and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used, that is, the highest safedose according to sound medical judgment. It will be understood by thoseof ordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of cancer. Alternatively, sustained continuous releaseformulations of the antibodies described herein may be appropriate.Various formulations and devices for achieving sustained release areknown in the art.

In one example, dosages for an antibody as described herein may bedetermined empirically in individuals who have been given one or moreadministration(s) of the antibody. Individuals are given incrementaldosages of the antibody. To assess efficacy of the antibody, anindicator of the disease (e.g., cancer) can be followed according toroutine practice.

Generally, for administration of any of the antibodies described herein,an initial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μs/kg to 3 μs/kg to 30 μs/kg to 300 μs/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate cancer, or a symptom thereof. An exemplary dosing regimencomprises administering an initial dose of about 2 mg/kg, followed by aweekly maintenance dose of about 1 mg/kg of the antibody, or followed bya maintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Forexample, dosing from one-four times a week is contemplated. In someembodiments, dosing ranging from about 3 μs/mg to about 2 mg/kg (such asabout 3 μs/mg, about 10 μs/mg, about 30 μg/mg, about 100 μs/mg, about300 μs/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the antibody used) can vary over time.

For the purpose of the present disclosure, the appropriate dosage of anantibody described herein will depend on the specific antibody (orcompositions thereof) employed, the type and severity of the cancer,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theadministration of the antibodies described herein may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced dose, e.g., either before, during, or after developing cancer.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has cancer, a symptom of cancer, or a predisposition towardcancer, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect cancer, the symptom of cancer, orthe predisposition toward cancer.

Alleviating cancer includes delaying the development or progression ofcancer, or reducing cancer severity. Alleviating cancer does notnecessarily require curative results. As used therein, “delaying” thedevelopment of cancer means to defer, hinder, slow, retard, stabilize,and/or postpone progression of cancer. This delay can be of varyinglengths of time, depending on the history of cancer and/or individualsbeing treated. A method that “delays” or alleviates the development ofcancer, or delays the onset of cancer, is a method that reducesprobability (the risk) of developing one or more symptoms of cancer in agiven time frame and/or reduces extent of the symptoms in a given timeframe, when compared to not using the method. Such comparisons aretypically based on clinical studies, using a number of subjectssufficient to give a statistically significant result.

“Development” or “progression” of cancer means initial manifestationsand/or ensuing progression of cancer. Development of cancer can bedetectable and assessed using standard clinical techniques as well knownin the art. However, development also refers to progression that may beundetectable. For purpose of this disclosure, development or progressionrefers to the biological course of the symptoms. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of cancer includes initial onset and/or recurrence.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

Diagnostic Applications

Described herein is a method for diagnosing cancer in a subject,comprising (a) applying a composition that includes one or moremonoclonal antibodies that detect expression of a panel of markersconsisting of GM3, GM2, GM1, GD1, GD1a, GD3, GD2, GT1b, A2B5, LeX, sLeX,LeY, SSEA-3, SSEA-4, Globo H, TF, Tn, sTn, CD44, CD24, CD45, CD90, CD133to a cell or tissue sample obtained from the subject; (b) assaying thebinding of the monoclonal antibody to the cell or the tissue sample; and(c) comparing the binding with a normal control to determine thepresence of the cancer in the subject.

Examples of the cancer for detection and diagnosis include, but are notlimited to, brain cancer, lung cancer, breast cancer, oral cancer,esophagus cancer, stomach cancer, liver cancer, bile duct cancer,pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovarycancer and prostate cancer. In some embodiments, the cancer is braincancer, lung cancer, breast cancer, ovarian cancer, prostate cancer,colon cancer, or pancreas cancer.

In some embodiments, the markers consist of GM2, GM1, GD1a, GT1b, A2B5,Tf, Tn, Globo H, SSEA3, SSEA4, CD24, CD44 and CD90. In some embodiments,the composition includes a plurality of monoclonal antibodies capable ofdetecting GM2, GM1, GD1a, GT1b, A2B5, Tf, Tn, Globo H, SSEA3, SSEA4,CD24, CD44 and CD90.

In some embodiments, the antibody is capable of detecting Globo H,SSEA-3 and SSEA-4-expressing cancer cells. In some embodiments, theantibody is capable of detecting Globo H and SSEA on cancer cells. Insome embodiments, the antibody is capable of detecting SSEA in cancers.In some embodiments, the cancer is brain cancer or glioblastomamultiforme (GBM) cancer, and the antibody is an anti-SSEA-4 monoclonalantibody.

Globo H, SSEA-3 and/or SSEA-4-specific monoclonal antibodies can be usedalone or in combination for in vitro and in vivo diagnostic assays todetect Globo H, SSEA-3 and SSEA-4-expressing cancer cells (e.g., GBM,certain solid tumor cells, and hematopoietic cancer cells as indicatedherein). For example, a sample (e.g., blood sample or tissue biopsy) canbe obtained from a patient and contacted with a triple-targetingantibody against Globo H, SSEA-3 and SSEA-4, or a GloboH/SSEA-3dual-targeting antibody in combination with an anti-SSEA-4, andthe presence of Globo H, SSEA-3 and SSEA-4 expressing cancer cells inthe patient sample can be determined by detecting antibody binding.Antibody binding can be detected directly (e.g., where the antibodyitself is labeled) or by using a second detection agent, such as asecondary antibody. The detectable label can be associated with anantibody of the invention, either directly, or indirectly, e.g., via achelator or linker.

In some embodiments, Globo H, SSEA-3 and/or SSEA-4 specific monoclonalantibodies are contacted with a biological sample from an individualhaving or suspected of having cancer, and antibody binding to a cell inthe sample is determined when higher or lower than normal antibodybinding indicates that the individual has cancer. In some embodiments,the biological sample is a blood sample or blood fraction (e.g., serum,plasma, platelets, red blood cells, white blood cells). In someembodiments, the biological sample is a tissue sample (biopsy), e.g.,from a suspected tumor site, or from a tissue that is known to beaffected, e.g., to determine the boundaries of a known tumor. In someembodiments, the biological sample is obtained from a site ofinflammation.

Biopsies are typically performed to obtain samples from tissues, i.e.,non-fluid cell types. The biopsy technique applied will depend on thetissue type to be evaluated (e.g., breast, skin, colon, prostate,kidney, lung, bladder, lymph node, liver, bone marrow, airway or lung).In the case of a cancer the technique will also depend on the size andtype of the tumor (e.g., solid, suspended, or blood), among otherfactors. Biopsy techniques are discussed, for example, in Harrison'sPrinciples of Internal Medicine, Kasper, et al., eds., 16th ed., 2005,Chapter 70, and throughout Part V.

Any method of detecting antibody binding to a cell in a sample can beused for the present diagnostic assays. Methods of detecting antibodybinding are well known in the art, e.g., flow cytometry, fluorescentmicroscopy, ELISAs, etc. In some embodiments, the method comprisespreparing the biological sample for detection prior to the determiningstep. For example, a subpopulation of cells (e.g., white blood cells)can be separated from the rest of the sample from the individual (e.g.,other blood components) or cells in a tissue can be suspended for easierdetection.

In some embodiments, the percentage of Globo H/SSEA-3/SSEA-4 expressingcells in the sample is determined and compared to a control, e.g., asample from an individual or group of individuals that are known to havecancer (positive control) or from an individual or group of individualsthat are known not to have cancer (normal, non-disease, or negativecontrol). In some embodiments, the control is a standard range of GloboH/SSEA-3/SSEA-4 expression established for a given tissue. A higher orlower than normal percentage of Globo H/SSEA-3/SSEA-4 expressing cells,or higher or lower expression level, indicates that the individual hascancer.

In one embodiment, a kit is provided for detecting Globo H, SSEA-3 andSSEA-4 in a biological sample, such as a blood sample or tissue sample.For example, to confirm a cancer diagnosis in a subject, a biopsy can beperformed to obtain a tissue sample for histological examination.Alternatively, a blood sample can be obtained to detect the presence ofGlobo H, SSEA-3 and SSEA-4. Kits for detecting a polypeptide willtypically comprise one or more antibodies that specifically bind GloboH, SSEA-3 and SSEA-4, such as any of the antibodies disclosed herein. Ina further embodiment, the antibodies are labeled (for example, with afluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of one or more antibodies that specifically bind Globo H,SSEA-3 and SSEA-4. The instructional materials may be written, in anelectronic form (such as a computer diskette or compact disk) or may bevisual (such as video files). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), enzyme linked immunosorbent assays (ELISA), orimmunohistochemical assays. The antibodies can also be used forfluorescence activated cell sorting (FACS). A FACS employs a pluralityof color channels, low angle and obtuse light-scattering detectionchannels, and impedance channels, among other more sophisticated levelsof detection, to separate or sort cells (see U.S. Pat. No. 5,061,620).Any of the monoclonal antibodies that bind to Globo H, SSEA-3 andSSEA-4, as disclosed herein, can be used in these assays. Thus, theantibodies can be used in a conventional immunoassay, including, withoutlimitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Westernblot or immunoprecipitation.

Methods for Staging and/or Determining Prognosis of Tumors

Another aspect of the present disclosure features a method for stagingand/or determining prognosis of tumorsin a human patient, the methodcomprising: (a) applying a composition that includes one or moreantibodies that detect the expression of markers consisting of SSEA-3,SSEA-4 and Globo H to a cell or tissue sample obtained from the patient;(b) assaying the binding of the monoclonal antibodies to the cell or thetissue sample; (c) comparing the expression level of the markers in thetest sample with the level in a reference sample, and (d) determiningthe stage and/or prognosis of tumors in the patient based upon theoutcome identified in step (c).

In some embodiments, the cancer is brain cancer, lung cancer, breastcancer, ovarian cancer, prostate cancer, colon cancer, or pancreascancer. In some preferred embodiments, the cancer is brain cancer orGBM.

In some embodiments, the antibody is capable of detecting Globo H,SSEA-3 and SSEA-4 expressing cancer cells. In some embodiments, theantibody is capable of detecting Globo H and SSEA on cancer cells. Insome embodiments, the antibody is capable of detecting SSEA in cancers.In some embodiments, the cancer is brain cancer or glioblastomamultiforme (GBM) cancer, and the antibody is an anti-SSEA-4 monoclonalantibody. In some embodiments, the antibody is an anti-SSEA-4 when thecancer is brain cancer or GBM.

In some embodiments, the provided glycan conjugates, immunogeniccompositions are useful in treating, or diagnosing a cancer, including,but are not limited to, acoustic neuroma, adenocarcinoma, adrenal glandcancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma,lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benignmonoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma),bladder cancer, breast cancer (e.g., adenocarcinoma of the breast,papillary carcinoma of the breast, mammary cancer, medullary carcinomaof the breast), brain cancer (e.g., meningioma; glioma, e.g.,astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer,carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma),choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g.,colon cancer, rectal cancer, colorectal adenocarcinoma), epithelialcarcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma,multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g.,uterine cancer, uterine sarcoma), esophageal cancer (e.g.,adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewingsarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM),a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronicmyelocytic leukemia (CIVIL), chronic neutrophilic leukemia (CNL),hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g.,neurofibromatosis (NF) type 1 or type 2, schwannomatosis),neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrinetumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g.,cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g.,pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm(IPMN), islet cell tumors), penile cancer (e.g., Paget's disease of thepenis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT),prostate cancer (e.g., prostate adenocarcinoma), rectal cancer,rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamouscell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cellcarcinoma (BCC)), small bowel cancer (e.g., appendix cancer), softtissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma,malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma,fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat glandcarcinoma, synovioma, testicular cancer (e.g., seminoma, testicularembryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of thethyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer),urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's diseaseof the vulva). In certain embodiments, the provided glycan conjugates,immunogenic compositions or vaccines are useful for treating braincancer, lung cancer, breast cancer, oral cancer, esophagus cancer,stomach cancer, liver cancer, bile duct cancer, pancreas cancer, coloncancer, kidney cancer, bone cancer, skin cancer, cervix cancer, ovarycancer, and prostate cancer.

To perform the treatment methods described herein, an effective amountof any of the glycan compositions described herein may be administeredto a subject in need of the treatment via a suitable route, as describedabove. The subject, such as a human subject, can be a patient havingcancer, suspected of having cancer, or susceptible to cancer. In someembodiments, the amount of the glycan conjugate or immunogeniccomposition is sufficient to elicit responses leading to the inhibitionof cancer growth and/or reduction of tumor mass. In other embodiments,the amount of the glycan composition may be effective in delaying theonset of the target cancer or reducing the risk for developing thecancer. The exact amount of the provided glycan compositions required toachieve an effective amount will vary from subject to subject,depending, for example, on species, age, and general condition of asubject, severity of the side effects or disorder, identity of theparticular compound(s), mode of administration, and the like. Thedesired dosage can be delivered three times a day, two times a day, oncea day, every other day, every third day, every week, every two weeks,every three weeks, or every four weeks. In certain embodiments, thedesired dosage can be delivered using multiple administrations (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations).

In certain embodiments, an effective amount, of the provided glycancompositions for administration one or more times a day to a 70 kg adulthuman may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg toabout 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg,about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg toabout 1000 mg, or about 100 mg to about 1000 mg, of a compound per unitdosage form.

In certain embodiments, the provided glycan compositions may beadministered orally or parenterally at dosage levels sufficient todeliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kgto about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg,preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kgto about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and morepreferably from about 1 mg/kg to about 25 mg/kg, of subject body weightper day, one or more times a day, to obtain the desired therapeuticeffect.

It will be appreciated that dose ranges as described herein provideguidance for the administration of the provided glycan conjugates,immunogenic compositions or vaccines to an adult. The amount to beadministered to, for example, a child or an adolescent can be determinedby a medical practitioner or person skilled in the art and can be loweror the same as that administered to an adult.

It will be also appreciated that the provided glycan compositions can beadministered in combination with one or more additional therapeuticallyactive agents. The provided glycan conjugates, immunogenic compositionsor vaccines can be administered in combination with additionaltherapeutically active agents that improve their bioavailability, reduceand/or modify their metabolism, inhibit their excretion, and/or modifytheir distribution within the body. It will also be appreciated that thetherapy employed may achieve a desired effect for the same disorder,and/or it may achieve different effects.

The provided glycan compositions can be administered concurrently with,prior to, or subsequent to, one or more additional therapeuticallyactive agents. In general, each agent will be administered at a doseand/or on a time schedule determined for that agent. In will further beappreciated that the additional therapeutically active agent utilized inthis combination can be administered together in a single composition oradministered separately in different compositions. The particularcombination to employ in a regimen will take into account compatibilityof the inventive compound with the additional therapeutically activeagent and/or the desired therapeutic effect to be achieved. In general,it is expected that additional therapeutically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

In certain embodiments, the provided glycan composition is administeredin combination with one or more additional pharmaceutical agentsdescribed herein. In certain embodiments, the additional pharmaceuticalagent is an anti-cancer agent. Anti-cancer agents encompassbiotherapeutic anti-cancer agents as well as chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limitedto, interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) andantibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR(tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to,anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRHagonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamideand bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A(2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (Abraxane),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765,AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523,PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIM1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTORinhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)),oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycinaminopterin, and hexamethyl melamine.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1: Exemplary Structure of Optimized Universal Fc Glycan

The glycan structure of an optimized universal Fc glycan for therapeuticantibodies is Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ (see FIG. 1).

Example 2: Exemplary General Procedure for the Preparation ofHomogeneous Antibodies with the Optimized Universal Glycan at the FcRegion

The present disclosure provides exemplary improved method for making apopulation of homogeneous antibodies with the optimized universal glycanat the Fc region comprising the steps of (a) contacting a monoclonalantibody with an α-fucosidase and at least one endoglycosidase, therebyyielding a defucosylated antibody having a single N-acetylglucosamine(GlcNAc), and (b) adding the universal glycan to GlcNAc of Fc region ofantibody to form the homogeneous antibody with the FIG. 1 showedoptimized glycan form (FIG. 2).

See FIG. 2. General strategy for the preparation of homogeneous antibodywith optimized universal glycan at the Fc region for the improvement ofits therapeutic activity.

Endoglycosidase is used to trim off the variable portions of anoligosaccharide in N-glycan. Examples of endoglycosidases used hereininclude, but not limited to, EndoA, EndoF, EndoF1, EndoF2, EndoH, EndoM,EndoS, and variants thereof.

Example 3: Preparation of Homogeneous Antibody with Universal Glycan atthe Fc Region Toward Enhancing Monoclonal Antibody Mediated AntiviralTherapeutics

Exemplary method for the preparation of homogeneous anti-influenza virusantibody with universal glycan at the Fc region to increase the its ADCCeffect.

Broadly neutralizing monoclonal antibodies targeting the conserved stalkregion of hemagglutinin (HA) can be facilitated by the interactionsbetween the antibody Fc and Fc receptors to trigger its function. Theanti-influenza virus antibody FI6 was chosen based on its demonstratedADCC effects, and the other anti-influenza virus antibody F10 thattarget stalk region of (HA) for the preparation of homogeneous antibodywith optimized universal glycan by using the general strategy andmethods disclosed herein. In brief, FI6 and F10 antibodies were preparedby the literature reported methods (ref). The home-made heterogeneousmonoclonal antibodies FI6 or F10 was used as the starting material andmodified with endoglycosidase endo S to yield a mixture of di-sugar mAbof GlcNAc-Fuc, and mono-sugar mAb of GlcNAc. Subsequently a homogeneousmono-sugar mAb was obtained with application of fucosidase; or themono-sugar species was obtained with combination of Endo S andfucosidase in one step.

FI6/F10 (0.25 mg) in a sodium phosphate buffer (50 mM, pH 7.0, 0.125 mL)was incubated with Endo S (12.5 μg) and BfFucH (0.25 mg) at 37° C. for22 h. LC-MS and SDS-PAGE analyses indicated the complete cleavage of theN-glycans on the heavy chain. The reaction mixture was subject toaffinity chromatography on a column of protein A-agarose resin (0.1 mL)that was pre-equilibrated with a sodium phosphate buffer (20 mM, pH7.0). The column was washed with a sodium phosphate buffer (20 mM, pH7.0, 1.0 mL). The bound IgG was released with glycine-HCl (50 mM, pH3.0, 1.0 mL), and the elution fractions were immediately neutralizedwith Tris-Cl buffer (1.0 M, pH 8.3). The fractions containing the Fcfragments were combined and concentrated by centrifugal filtration(Amicon Ultra centrifugal filter, Millipore, Billerica, Mass.) to givemono-GlcNAc homogeneous antibody (0.193 mg). The product wastrypsinized, and the glycopeptides, TKPREEQYNSTYR and EEQYNSTYR, wereanalyzed using nanospray LC/MS to confirm the glycosylation pattern ofglycan engineering FI6/F10.

Isolation of the sialylglycan (SCT) from hen's egg yolk was according tothe published method with some modification. Briefly, the ethanolextraction of hen's egg yolk was centrifuged, filtrated, and the treatedwith endo M, after reaction complete, the SCT was purified by gelfiltration and ion exchange chromatography, the purified SCT waslyophilized to give pure SCT product as a white powder (82%).

A solution of SCT (Sia₂Gal₂GlcNAc₂Man₃GlcNAc) (3.0 mg),2-chloro-1,3-dimethylimidazolinium chloride (DMC) (6.3 mg) and Et₃N (9.0μL) in water (60.0 μL) was stirred at 4° C. for 1 h. The reactionmixture was subjected to gel filtration chromatography on a SephadexG-25 column eluted by 0.05% aqueous Et₃N. The fractions containing theproduct (SCT oxazoline) were combined and lyophilized to give a whitepowder (2.6 mg, yield 87.4%).

SCT oxazoline was added to a mixture of glycosynthase and mono-GlcNAcFi6 or F10 in 50 mM Tris buffer (pH 7.8) and incubated for an hour atroom temperature. The reaction mixture was purified with protein Aaffinity column, followed by amanion exchange column capto Q to collectthe desired product, optimized anti-influenza virus homogeneous antibodyFI6-M or F10-M. The product was trypsinized, and the glycopeptides,TKPREEQYNSTYR and EEQYNSTYR, were analyzed using nanospray LC/MS toconfirm the glycosylation pattern of FI6-M or F10-M.

Example 4

ADCC assay of FI6/F10 and glycoengineering FI6-M/F10-M. See FIG. 3demonstrated the enhanced ADCC results of anti-influenza virusantibodies.

Anti-viral antibody-dependent cell-mediated cytotoxicity (ADCC)enhancement is demonstrated with anti-influenza monoclonal antibodiesFI6 and F10 with glycan modification. Human HEK293T cells weretransiently transfected with plasmid to express full-length Cal/09 HA onthe cell surface to mimic influenza virus infected cells. These cellswere mixed with freshly prepared human peripheral blood mononuclearcells (PBMC) isolated from health donors with ratio of infected cells toPBMC of 1:20 or 1:50. Antibodies FI6 and F10 in different concentrationswith and without glycan modification are then added into the mixtures.After 5 hours, the result of FI6 and F10 induced ADCC was monitored byHEK293T cell lysis (LDH release). The results show that the ADCC inducedby antibodies FI6 and F10 with glycan modification is enhanced 1.5-3folds.

Example 5: Exemplary Methods and Materials for ADCC Assay ExampleAnti-Stem Monoclonal Antibodies FI6 and F10

The F10 and FI6 antibody expression plasmids were transfected to HEK293Fcell by using polyethyleneimine and cultured in Freestyle 293 expressionmedium (Invitrogen). After 7 days incubation, the supernatants werecollected by centrifugation and the antibodies were purified by proteinA beads (Roche Diagnostics). The antibody was further purified by gelfiltration chromatography on Superdex 200 (GE Healthcare) in PBS buffer.

Example 6: In Vitro Antibody-Dependent Cellular Cytotoxicity (ADCC)Assay

HEK293T cells were transfected with pVax-Cal/09 hemagglutinin (HA)expression plasmid for 48 hour. The HA-expressing HEK293T cells weretrypsinized and seeded in 96-well U-bottom plates, 5,000 cells per wellin 50 ul DMEM medium (Gibco).

Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Paqueseparation of whole blood obtained from healthy volunteers and used aseffector cells in the ADCC assay. Briefly, whole blood was diluted withan equal volume of HBSS, layered over Ficoll-Paque plus (GE Healthcare)and centrifuged at 400 g for 40 min. The PBMC cells were harvested,washed twice with HBSS and mixed with HA-expressing HEK293T cells usingan effector-to-target ratio of 50/1.

Mixture of PBMCs and HA-expressing HEK293T cells were treated withdifferent concentrations of antibodies FI6 and F10 and incubated at 37°C. for 5 hours.

After 5 hour incubation, ADCC was monitored by measuring the lactatedehydrogenase (LDH) released using cytoTox96 Non-RadioactiveCytotoxicity Assay kit (Promega).

Example 7: Preparation of Homogeneous Antibody with Universal Glycan atthe Fc Region Toward Enhancing Monoclonal Antibody Mediated Anti-CancerTherapeutics Representative Examples

Commercial available Rituxan® and Herceptin® were used as startingmaterial, after the same methods described previously for thepreparation of homogeneous anti-influenza virus antibody with universalglycan at the Fc region. The homogeneous Rituxan® and Herceptin® withthe optimized universal glycan Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ at theFc region can be obtained. Using the same methods, we have also prepareddifferent homogeneous Rituxan® and Herceptin® antibodies with differentglycanform at their Fc region for the comparison of antibodiesactivities with different glycans.

Biological Characteristic of Anti-CD20 Homogeneous Antibody

Glycosylation on Fc can affect a variety of immunoglobulineffector-mediated functions, including ADCC, CDC and circulatinghalf-life. ADCC enhancement is a key strategy for improving therapeuticantibody drug efficacy. It can lowering effective drug dosage forbenefits of lower drug cost. The anti-CD20 homogeneous antibodiesdescribed herein can be characterized by functional properties. Theanti-CD20 GAb has cell growth inhibitory activities including apoptosisagainst human CD20 expressing cells. In some embodiments, the anti-CD20GAb exhibits more potent cell growth inhibitory activities as comparedto its patent antibody.

Example 8: ADCC Activities of Anti-CD20 Glycoantibodies

The ADCC activity of the homogeneous antibody according to the inventionis at least 8 fold increased, preferably at least 15 fold, morepreferably at least 35 fold increased ADCC activity, preferably at least50 fold increased ADCC activity, preferably at least 60 fold increasedADCC activity, most preferred at least 80 fold increased ADCC activitycompared to the ADCC activity of the parental antibody.

The ADCC lysis activity of the inventive homogeneous antibody can bemeasured in comparison to the parental antibody using target cancer celllines such as, for example, SKBR5, SKBR3, LoVo, MCF7, OVCAR3 and/or KatoIII.

A number of anti-CD20 GAbs described herein, in particular GAb101, andGAb104, exhibited enhanced ADCC activity compared to it parentalantibody, Rituximab. The homogeneous antibodies of the invention canexhibit superior effect as therapeutic agents for B cell-mediatedmalignant tumors and immunological diseases in which B cells orantibodies produced by B cells are involved, and an object of thepresent invention is to use the anti-CD20 GAb in development oftherapeutic agents.

Example 9: CDC Activities of Anti-CD20 Glycoantibodies

The homogeneous antibodies described herein are surprisingly able toprovide improved ADCC without affecting CDC. Exemplary CDC assays aredescribed in the examples. In exemplary embodiments, ADCC of theglycoantibody is increased but other immunoglobulin-type effectorfunctions such as complement-dependent cytoxicity (CDC) remain similaror are not significantly affected.

Binding Between FcγRIII and Anti-CD20 Glycoantibodies

FcγRIIIA was transfected into HEK-293 cell line to express recombinantprotein. The secreted FcγRIIIA recombinant protein was purified and thendiluted to serial concentration in HBS-EP buffer (200 nM, 100 nM, 50 nM,25 nM, and 12.5 nM). Each of anti-CD20 GAbs101, 102, 104, 105, 106, 107,108, 109, 110 and 111 was diluted in HBS-EP buffer to the concentrationof 10 mg/ml, and then captured to the CM5 chip in which anti-human Fabdomain antibodies were pre-immobilized. A serial titration of FcγRIIIAwas injected and bound at the flow rate of 30 ml/min. Single cyclekinetics data was fitted into 1:1 binding model using Biacore T200evaluation software to measure the equilibrium constant (Ka/Kd). Theresults were shown in Table 2.

Table 2 lists exemplary FcγRIIIA binding of anti-CD20 GAbs andRituximab. FcγRIIIA binding may be measured using assays known in theart. Exemplary assays are described in the examples. The Fc receptorbinding may be determined as the relative ratio of anti-CD20 GAb vsRituximab. Fc receptor binding in exemplary embodiments is increased byat least 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 15-fold or 20-fold, 30-fold, 40-fold, 50-fold,100-fold or higher.

Table 2.

As compared to Rituximab, the binding data showed that the anti-CD20GAbs, in particular GAb101 and GAb104, exhibit stronger binding affinityfor the target molecule CD20.

Taken together, anti-CD20 GAbs, in particular GAb101, and GAb104,exhibited enhanced ADCC activity and stronger FcγRIIIA binding affinityas compared to Rituximab. The homogeneous antibodies of the inventioncan provide a superior clinical response either alone or, preferably, ina composition comprising two or more such antibodies, and optionally incombination with other treatments such as chemotherapy. TheADCC-enhanced anti-CD20 glycoantibody can provide an alternativetherapeutic for B-cell lymphoma and other diseases. The glycoantibodiesof the present invention can be used to alter current routes ofadministration and current therapeutic regimens, as their increasedeffector function means they can be dosed at lower concentrations andwith less frequency, thereby reducing the potential for antibodytoxicity and/or development of antibody tolerance. Furthermore, theirimproved effector function yields new approaches to treating clinicalindications that have previously been resistant or refractory totreatment with the corresponding anti-CD20 monoclonal antibody producedin recombinant host systems.

Example 10: Binding to B-Lymphoma Cells

The binding activities of Rituxan-SCT (GAb101) and Rituxan mono-GlcNActo Ramos cells, Raji and SU-DHL-4 cells were examined, and the resultsshowed both have similar binding activities as Rituximab (FIG. 4).

FIG. 4. Binding activities of different homogeneous antibodies withdifferent cells with CD20.

Example 11: CDC to B-Lymphoma Cells

The CDC effects of Rituxan-SCT (GAb101) and Rituxan mono-GlcNAc to Ramoscells, Raji and SU-DHL-4 cells were tested. The comparative CDC profilesseen with Ramos cells (enhanced by GAb101 and reduced by Riruxan-GlcNAc)were confirmed in the other B-lymphoma cell line SU-DHL-4 (FIG. 5 rightpanel). Reproducible results were obtained when conducted on a secondoccasion using different cell passages.

Example 11

See FIG. 5. Depletion of human B cells

The depletion of human B cells was conducted using human PBMC cellsfreshly prepared from human blood. The cells at 2×10⁶ in RPMI 1640-5%FBS cultured on microplates were incubated, in the absence or presenceof 15% autologous plasma, at 37° C. for 4 hr with the anti-CD20 GAbsRituxan-SCT, Rituxan-GlcNAc and Rituximab at different concentrations.The cells after wash were stained with anti-CD2-PE and anti-CD19-FITC onice for 5 min. B cells depletion was analyzed on FACS, based on theCD19⁺ CD2⁻ B cells. (FIG. 6) See FIG. 6. Depletion of human B cells bydifferent homogeneous antibodies.

Example 12: Binding to B-Lymphoma Cells

The binding of the antibodies was investigated in CD20⁺ B lymphoma celllines (Ramos, Raji, and) and analyzed on flow cytometry. The cells inPBS containing 1% fetal bovine serum at 2×10⁵/well on microplate wereincubated on ice for 1 hr with antibodies of interest at differentconcentrations. The cells are washed, re-suspended in the PBS buffer,and incubated with the detecting goat anti-hIgG-Fcγ-PE on ice for 30min. The cells are washed and subjected to analysis on FACS.

Example 13: Binding to FcRI-IIIa-Expressing CHO Cells

The binding of the antibodies to the FcRIIIa receptors (CD16a), which isa precursor event known to be correlative with the induction ofantibody-dependent cellular cytotoxicity (ADCC), was investigated in CHOcells transfected with the high-affinity CD16a (158Val) and analyzed onflow cytometry. The cells in PBS containing 1% fetal bovine serum at1×10⁵/well on microplate were incubated on ice for 1 hr with antibodiesof interest at different concentrations. The cells are washed,re-suspended in the PBS buffer, and incubated with the detecting goatanti-hIgG-Fcγ-PE on ice for 30 min. The cells are washed and subjectedto analysis on FACS.

Complement-dependent cytotoxicity (CDC) to B-lymphoma cells. The CDCeffect induced by the antibodies were investigated in CD20+B lymphomacell lines (Ramos and SKW6.4) and analyzed on flow cytometry. The cellsin RPMI 1640 culture medium at 2.0×105/well on microplates wereincubated on ice for 30 min with antibodies of interest at differentconcentrations. The cells were washed and incubated at 37° C. for 30 minwith 10% human serum in RPMI 1640. The cells were washed and incubatedin the dark for 5 min with the PI reagent. The cell deaths by CDC wereanalyzed on FACS.

Antibody-dependent cellular cytotoxicity (ADCC) to B-lymphoma cells. TheADCC effect induced by the glyco-antibodies were investigated inCD20-containing B lymphoma cell lines (Ramos and SKW6.4), using freshlyprepared human PBMC as effector cells, and the results analyzed on flowcytometry. The target B cells in PBS-0.1% BSA were first labeled withCFSE at 37° C. for 5 min. After wash the CFSE-labeled cells in RPMI 1640medium were incubated at 37° C. for 4 hr on microplates with theglyco-antibodies of interest at different concentrations and PBMCeffector cells. The ratio of target cells to effector cells was set at25:1. The resultant mixtures were stained in the dark for 5 min with thePI reagent. The cell deaths by ADDC were analyzed on FACS.

Depletion of human B cells. The depletion of human B cells was conductedusing human PBMC cells freshly prepared from human blood. The cells at2×106 in RPMI 1640-5% FBS cultured on microplates were incubated, in theabsence or presence of 15% autologous plasma, at 37° C. for 4 hr withthe antibodies of interest at different concentrations. The cells afterwash were stained with anti-CD2-PE and anti-CD19-FITC on ice for 5 min.B cells depletion was analyzed on FACS, based on the CD19+CD2-B cells.

Preparation of Homogeneous Herceptin® by the Strategy of GlycanEngineering.

Methods to prepare different glycan modified homogeneous Herceptin®.

Biological Characteristic of Anti-HER2 Homogeneous Antibodies

Glycosylation on Fc can affect a variety of immunoglobulineffector-mediated functions, including ADCC, CDC and circulatinghalf-life. ADCC enhancement is a key strategy for improving therapeuticantibody drug efficacy. It has the potential of lowering effective drugdosage for benefits of lower drug cost. The anti-HER2 glycoantibodiesdescribed herein can be characterized by functional properties. Theanti-HER2 GAb has cell growth inhibitory activities including apoptosisagainst human HER2 expressing cells. In some embodiments, the anti-HER2GAb exhibits more potent cell growth inhibitory activities as comparedto its patent antibody.

ADCC Activities of Anti-HER2 Glycoantibodies

The ADCC activity of the glycoantibody according to the invention is atleast 3 fold increased, preferably at least 9 fold, more preferably atleast 10 fold increased ADCC activity, preferably at least 12 foldincreased ADCC activity, preferably at least 20 fold increased ADCCactivity, most preferred at least 30 fold increased ADCC activitycompared to the ADCC activity of the parental antibody.

The ADCC lysis activity of the inventive glycoantibody can be measuredin comparison to the parental antibody using target cancer cell linessuch as SKBR5, SKBR3, LoVo, MCF7, OVCAR3 and/or Kato III.

Table 3 lists exemplary enhanced ADCC activities of anti-HER2 GAbs ascompared to Trastuzumab. Exemplary assays are described in the examples.

TABLE 3 Anti-HER2 Trastuzumab GAb101 GAb104 GAb105 GAb107 GAb108 GAb111ADCC 1 30 14.3 9.5 10 6.5 3 (fold)

A number of anti-HER2 GAbs described herein, in particular GAb101, andGAb104, exhibit enhanced ADCC activity compared to it parental antibody,Rituximab. It is contemplated that the glycoantibodies of the inventionmay exhibit superior effect as therapeutic agents for HER2-positivediseases, and an object of the present invention is to use the anti-HER2GAb in development of therapeutic agents.

CDC Activities of Anti-HER2 Glycoantibodies

The glycoantibody described herein is surprisingly able to provideimproved ADCC without affecting CDC. Exemplary CDC assays are describedin the examples. In exemplary embodiments, ADCC of the glycoantibody isincreased but other immunoglobulin-type effector functions such ascomplement-dependent cytoxicity (CDC) remain similar or are notsignificantly affected.

Binding Between FcγRIII and Anti-HER2 Glycoantibodies

Table 4 lists exemplary FcγRIIIA binding of anti-HER2 GAbs andRituximab. Table 4.

FcγRIIIA binding may be measured using assays known in the art.Exemplary assays are described in the examples. The Fc receptor bindingmay be determined as the relative ratio of anti-HER2 GAb vs Trastuzumab.Fc receptor binding in exemplary embodiments is increased by at least2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 15-fold or 20-fold, 30-fold, 40-fold, 50-fold or higher.

As compared to Trastuzumab, the binding data showed that the anti-HER2GAbs, in particular GAb101 and GAb104, exhibit stronger binding affinityfor the target molecule HER2.

Taken together, anti-HER2 GAbs, in particular GAb101, and GAb104,exhibit enhanced ADCC activity and stronger FcγRIIIA binding affinity ascompared to Trastuzumab. It is contemplated that the glycoantibodies ofthe invention may provide a superior clinical response either alone or,preferably, in a composition comprising two or more such antibodies, andoptionally in combination with other treatments such as chemotherapy. Itis contemplated that the ADCC-enhanced anti-HER2 glycoantibody mayprovide an alternative therapeutic for HER2-positive diseases. Theglycoantibodies of the present invention advantageously can be used toalter current routes of administration and current therapeutic regimens,as their increased effector function means they can be dosed at lowerconcentrations and with less frequency, thereby reducing the potentialfor antibody toxicity and/or development of antibody tolerance.Furthermore, their improved effector function yields new approaches totreating clinical indications that have previously been resistant orrefractory to treatment with the corresponding anti-HER2 monoclonalantibody produced in recombinant host systems.

Preparation of homogeneous antibody with universal glycan (SCT) at theFc region toward enhancing monoclonal antibody mediatedanti-inflammation therapeutics

The Fc region with siaa2,6Gal structure can increase the activities ofanti-inflammation. Here we prepare the homogeneous Humira with SCTglycan at the Fc region to improve its anti-inflammation activities.

General Procedure for Analysis of N-Glycosylation of Anti-TNFα

We developed a mass spectrometric method to monitor the yield ofoligosaccharide-derived fragment ions (oxonium ions) over a collisioninduced dissociation (CID) energy applied to a glycopeptides precursor.Multiple Reaction Monitoring (MRM) of oxonium ions method could fulfillthe regulatory requirement on the routine quality control analysis offorthcoming biosimilar therapeutics.

5 ug of Adalimumab (Humira®) (purchased from Abbvie) was dissolved in 25ul of 2M Guanidine-HCl, and dithiothreitol (DTT) were added to a finalconcentration of 5 mM. After 10 minutes incubation in 110° C., reducedcysteine residues were alkylated in 10 mM Iodoacetamide (IAA) at 37° C.for 1 hour. Add 5 mM DTT to quench excess IAA at RT for 10 minutes. Theproduct was diluted 15 times in 50 mM ammonium bicarbonate beforemicrocentrifugation with spin column (10 kDa protein MW cut-off). Thetrypsin digestion was performed for 4 hours at 37° C. using an enzyme:protein ratio of 1:25 (w/w). Sample was frozen at −20° C. for LC-MS/MSanalysis.

Instrumentation

The glycopeptide quantification by m/z 204 oxonium ion (HexNAc)monitoring was performed using a 4000 QTrap triple quadrupole massspectrometer (AB Sciex) with Aglient 1200 HPLC system. For relativequantification of glycopeptide microheterogeneity, precursor ion m/z wasderived in-silico, covering all possible glycan compositions, and asingle quantitative transition was monitored for each precursor ion (Q3m/z=204).

MS Data Analysis

The acquired raw data was processed with Analyst 1.5 (AB Sciex). Themass chromatogram of each transition was integrated and quantified bypeak area. The percentage composition of each component was calculatedwith respect to the sum of all components combined.

Preparation of Anti-TNFα Antibody Humira-SCT

Isolation of the sialylglycopeptide (SGP) from hen's egg yolk wasaccording to the published method. Briefly, the phenol extraction ofhen's egg yolk was centrifuged, filtrated, and purified by thechromatographic columns, including Sephadex G-50, Sephadex G-25,DEAE-Toyoperarl 650M, CM-Sephadex C-25 and Sephadex G-25. A solution ofsialylglycopeptide (SGP) (52 mg) in a sodium phosphate buffer (50 mM, pH6.0, 5 mM) was incubated with the Endo M (53 μg) at 37° C. After 7 hour,the reaction mixture was subjected to gel filtration chromatography on aSephadex G-25 column eluted by water. The fractions containing theproduct were combined and lyophilized to give the product (glycan-101)as a white powder (30 mg, yield 82%).

A solution of glycan-101 (Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc) (30 mg),2-chloro-1,3-dimethylimidazolinium chloride (DMC) (62.7 mg) and Et₃N (89μL) in water was stirred at 4° C. for 1 h. The reaction mixture wassubjected to gel filtration chromatography on a Sephadex G-25 column andeluted by 0.05% aqueous Et₃N. The fractions containing the product (SCToxazoline) were combined and lyophilized to give a white powder.

SCT oxazoline was added to a mixture of endoglycosidase and GAbHumira-GlcNAc in 50 mM Tris buffer (pH 7.8) and incubated for an hour atroom temperature. The reaction mixture was purified with protein Aaffinity column, followed by amanion exchange column capto Q to collectthe desired product, anti-TNFα GAb101. The product was trypsinized, andthe glycopeptides, TKPREEQYNSTYR and EEQYNSTYR, were analyzed usingnanospray LC/MS to confirm the glycosylation pattern of Humira-SCT.

Binding Affinity of Anti-TNFα

Human recombinant TNF-α containing 158 amino acids (MW=17.5 kDa) wasproduced in E. coli (PROSPEC) and purified. Recombinant human TNF-αprotein was titrated and a serial dilution of 50 nM, 25 nM, 12.5 nM,6.25 nM, and 3.125 nM was prepared in HBS-EP buffer. Adalimumab andanti-TNFα GAb200 and 401 were diluted in HBS-EP buffer to aconcentration of 10 μg/ml, and then captured to the CM5 chip whereanti-human Fc domain antibodies were pre-immobilized. Serialconcentration of recombinant human TNF-alpha as the analyte and theninjected and bound to the captured antibody on chip at the flow rate of30 μl/min. After binding, the antibody-analyte complex were washed byregeneration buffer, 10 mM glycine-HCl pH1.5 at the flow rate of 50μl/min. CM5 chip was maintained in PBS pH7.4 at 4° C. for further use.Single cycle kinetics data was fitted into 1:1 binding model usingBiacore T200 evaluation software to measure the equilibrium constant(Ka/Kd).

Example 13: Generation of Anti-SSEA-4 Monoclonal Antibodies

Hybridoma methodology was employed for the development of mAbs specificto SSEA-4. Female BALB/c mice, aged 6-8 weeks old, were immunized threetimes subcutaneously with the SSEA-4 vaccine. Three immunizations weregiven at 2-wk intervals. Each vaccination contained 2 μg of SSEA-4. Allof the sera were obtained by centrifugation at 4,000×g for 10 min. Theserologic responses were analyzed by glycan microarray. A final boostwas given intraperitoneally with 2 μg of SSEA-4, and 3 days later, thespleen cells from immunized mice were used for generating hybridomas.

Hybridoma cells secreting antibodies with the desired antigen-bindingactivities were screened as follows. Microtiter plates were coated byincubating with 4 μg/mL of neutravidin in carbonate buffer, 0.1M, pH9.6, overnight at 4° C. The wells were blocked with 1% BSA in PBS,pH=7.3 for 1 hour and incubated with 4 μg/mL SSEA-4-biotin for 1 hour.The antisera were at various dilutions for 1 hour at 37° C. Afterwashing, the ligand-bound antibodies were detected by HRP-conjugatedgoat anti-mouse IgG or IgM antibody (Jackson ImmunoResearch) at 1:10,000and incubated for 1 hour at 37° C., followed by incubation with TMBsubstrate. The OD was determined at 450 nm. Positive clones wereselected for further characterization. Three exemplary clones 45, 46 and48, were identified in this study as specifically binding to SSEA-4. Formouse monoclonal isotyping, the IsoQuick Strips and Kits was used(sigma, 19535). Add hybridoma medium to the reaction vial. Insert thestrip into the sample making sure the strips are upright. The samplewill travel up the strip. Allow the strip to develop for 5 minutesbefore making final interpretations.

The V_(H) and V_(L) gene segments of the mAbs 45, 46 and 48 wereamplified by PCR from the hybridoma clone secreting the antibody. Thegene segments thus obtained were sequenced to determine the V_(H) andV_(L) sequences of mAbs 45, 46 and 48, which are shown in Tables 3-5.

Example 14: Generations of Chimeric Antibodies

The V_(H) and V_(L) gene segments of the mAb 273 and 651 were amplifiedby PCR from the hybridoma clone secreting the antibody. The genesegments thus obtained were sequenced to determine the V_(H) and V_(L)sequences of mAb 273 and 651, which are shown in Tables 1 and 2. Theheavy chain and light chain variable region were cloned to human IgG1antibody expression vector show as FIG. 9. VH was using enzyme siteBsiWI and ApaI, and VL was using enzyme site BsPEI and NheI Vectors weretransiently transfected into either 293F or CHO-S cells. Recombinantchimeric Ab was purified and further study for binding assay andcomplement-dependent tumor cell lysis assay.

The V_(H) and V_(L) gene segments of the mAb 46 and 48 were amplified byPCR from the hybridoma clone secreting the antibody. The gene segmentsthus obtained were sequenced to determine the V_(H) and V_(L) sequencesof mAb 46 and 48, which are shown in Tables 5 and 4. The heavy chain andlight chain variable region were cloned to human IgG1 antibodyexpression vector show as FIG. 9. VH was using enzyme site BsiWI andApaI, and VL was using enzyme site BsPEI and NheI Vectors weretransiently transfected into either 293F or CHO-S cells. Recombinantchimeric Ab was purified and further study for binding assay andcomplement-dependent tumor cell lysis assay.

Example 15: Binding Analysis of Antibodiesto Cancer Cells by FlowCytometry

Binding of mAb 273 and anti-SSEA-4 (mAbs 45, 46 and 48) to cancer celllines were examined. Cells (1×10⁵) were resuspended in 100 μL FACSbuffer (1% BSA/PBS solution) containing various concentration antibodyand incubated on ice for 30 min. After being washed twice with FACSbuffer, cells were incubated with 649-labeled goat anti-mouse antibody(1:100; Jackson ImmunoResearch) for 30 min on ice before analysis on aFACSCalibur system (BD Biosciences). The results are shown in FIGS.7A-D. Breast cancer cells MCF-7 were stained with mAb 273 (FIG. 7A).Pancreatic cancer cells (HPAC and BxPC3) and breast cancer cells MCF-7were stained with mAb 45 (FIG. 7B). Pancreatic cancer cells (HPAC andBxPC3) and breast cancer cells MCF-7 were stained with mAb 46 (FIG. 7C).Pancreatic cancer cells (HPAC and BxPC3) and breast cancer cells MCF-7were stained with mAb 48 (FIG. 7D).

We also used the glycan array to determine the dissociation constants ofMC45, MC48 and MC813-70 with SSEA-4 hexasaccharide on surface, and theKd values for MC45, 48 and 813 are shown below. These results showedthat these mAbs are highly specific for SSEA4.

Kd (nM) ± SD(nM) MC45 0.37 ± 0.08 MC48 0.46 ± 0.1  MC813-70 4.21 ± 0.26

Example 16

The ability of exemplary mAbs 46 and 48 to mediate CDC of SSEA-4expressing cells was examined. Homo sapiens pancreas adenocarcinoma cell(BxPC3) in the presence of rabbit serum as a source of complement. Celldeath was assessed by the addition of the viability probe 7-AAD. Basedon the results of the 7-AAD measurement, percentage-specific lysis wascalculated using a FACScan flow cytometer. The antibodies showed about20% killing activity at 40 μg/mL. As shown in FIG. 5(C), mAbs 46 and 48successfully mediated CDC of SSEA-4 expressing cells.

Example 15: Exemplary Phage Display Biopanning Procedures

The phage-displayed human naïve scFv library contained 2.5×10¹⁰ clones(Lu et al., 2011) was subtracted with non-specific binding inPEG-conjugated carboxyl Dynabeads (Invitrogen) at room temperature (RT)for 1 hour, and subsequently incubated with SSEA-4-PEG immobilizedDynabeads at 4° C. for 1 hour. After washing with PBS or PBS containing0.01% Tween 20 (PBST0.01), the phages that bound to SSEA-4-PEG-Dynabeadswere recovered by infection with E-coli TG1 cells at 37° C. for 0.5hour. Some of the infected cells were serially diluted to determinetiter, and the others were rescued by M13KO7 phage and amplified. Afterdetermination of rescued phages titer, the next round of biopanning wasperformed. In the fourth and fifth round of biopanning, the phage cloneswere randomly selected to culture for ELISA screening.

ELISA Screening of Selected Phage Clones

For detection of antigen recognition, microwell plates (Nunc) werecoated with 0.2 μg/ml of SSEA-4-BSA, Globo H-BSA, SSEA-3-BSA and BSA,respectively. The selected phage clones were diluted 1:2 in PBScontaining 3% BSA and added to each well. The plates were incubated atRT for 1 hour, washed with PBST0.1, and incubated with horseradishperoxidase (HRP)-conjugated mouse anti-M13 phage antibody (GEHealthcare). The plates were washed again, and OPD and H2O2 were added.After termination of reaction by 3 N HCl, the absorbance was measuredusing a 490 nm using microplate reader (Model 680, BioRad). We extractedphagemids from ELISA-positive phage clones to identity scFv codingregions by auto-sequencing.

Construction and Expression of Anti-SSEA-4 Human IgG

The VH region of selected scFv was cloned with AgeI and NheI site intomodified expression vector pcDNA5-FRT-Gamma1 containing a signal peptideand the constant region of human immunoglobulin gamma 1 heavy chain. TheVL region of selected scFv was cloned with AgeI and EcoRV site intomodified expression vector p-Kappa-HuGs containing a signal peptide andconstant region of human immunoglobulin kappa light chain. Both plasmidswere transfected into FreeStyle293 cells (Invitrogen) and continuouslyincubated in serum-free medium at 37° C. for 1 week to produce humanantibody.

Purification of Anti-SSEA-4 Human IgG

The culture medium was collected, centrifuged and filtrated with 0.45 μmpore-size membrane. The supernatant then was subjected to protein Gcolumn chromatography (GE healthcare) for purification of anti-SSEA-4human IgG. After dialysis of eluents with PBS, the antibody was examinedby SDS-PAGE analysis with coomassie blue staining as usual. Theconcentration of antibody was assessed by Bradford reagent (ThermoScientific) and spectrophotometer.

Humanization of MC48

Two human genes, GenBank accession Q9UL73 and AY577298, were the mostsimilar to MC48 VH and VL, respectively. We humanized three sequences ofMC48, including the 1st humanized MC48 (hMC48) VH consisted of modifiedframework (FR) 1 to FR4 of Q9UL73 gene and the 1st hMC48 VL consisted offour FRs from the accession AY577298, the 2nd hMC48 FRs of VH followed1YY8 from PDB, while the 2nd hMC48 VL same as 1st sequence, and the 3rdhMC48 VH sequence modified FR1, 2 and 4 of Q9UL73 gene and the 3rd hMC48VL changed FR2 and FR4 to human AY577298 gene. All of these humanizedsequences were conserved CDR1 to CDR3 of VH and VL of MC48.

Construction of Single Chain Fragments Variable (scFv) of Humanized MC48Variants

The scFv form of humanized MC48 sequences (VH-GGGGSGGGGSGGGGS-VL (SEQ IDNO: 115)) were gene synthesized (Genomics) and cut by Sfi I and Not I(Fermentas). After gel extraction, the digested products were cloned topCANTAB-5E phagemid (GE Healthcare).

Generation of Humanized MC48 (hMC48) scFv Phage Clones.

hMC48 variant phagemids were transformed to TG1 E-coli and recovered in2×YT medium (BD Pharmingen) containing 100 μg/ml ampicillin and 2%glucose and rescued by M13KO7 helper phage (NEB) for 1 hour at 37° C.After centrifugation by 1,500×g for 10 min, these pellets wereresuspended in 2×YT medium containing 100 g g/ml ampicillin and 50 μg/mlkanamycin overnight to generate scFv-phages.

Binding Assay of hMC48 scFv Phage Clones by ELISA

SSEA-4-BSA was coated on an ELISA plate at the concentration of 0.2μg/ml. After washing and blocking, the serial diluted phages wereincubated at RT for 1.5 hour. After washing, 1:1000 dilutedHRP-conjugated anti-M13 antibody (GE Healthcare) was added at RT for 1hour. Then, liquid substrate 3,3′,5,5′-tetramethylbenzidine (TMB)developed and was terminated with 3N HCl. Optical density was measuredat 450 nm.

Results

Identification of Phage-Displayed scFv that Binds to SSEA-4

To identify the antibodies that bind to SSEA-4, we used phage-displayedhuman naïve scFv library containing 2.5×10¹⁰ members which wasestablished as our previous report described (Lu et al., 2011). Thislibrary was first removed Dynabeads-binding phages and then selected forSSEA-4-binding phages by SSEA-4-PEG-conjugated Dynabeads. We used twobuffer systems, PBS and PBS containing 0.01% Tween20 (PBST0.01), duringbiopanning. After five rounds of affinity selection, the phage recoveryof the fifth round had increased about 55-fold and 80-fold than that ofthe first round in PBS and PBST0.01 system, respectively (FIG. 10). Thephage clones were randomly selected and tested for SSEA-4 binding byELISA (FIG. 11). We found seven clones that specifically bound toSSEA-4-BSA, but not to BSA control protein. By sequencing all 8individual clones, we identified two unique anti-SSEA-4 phage clones(p1-52 and p2-′78) which contain distinct human VH and VL coding regions(FIG. 16A).

To examine the specificity and binding affinity of the two phage clones,we performed a comparative ELISA using the same phage titer toGlobo-series glycans including SSEA-4-BSA, Globo H-BSA and SSEA-3-BSA(FIG. 12). The p2-78 phage clone showed the strong binding to SSEA-4-BSAand SSEA-3-BSA, and slightly weaker binding to Globo H-BSA. However, wefound that the binding activity of p1-52 phage clone to SSEA-4-BSA isvery weak. Thus we focused on p2-78 clone for further study.

To establish the fully human antibody (hAb) against SSEA-4, wemolecularly engineered the VH and VL coding sequences of p2-78 scFv intohuman IgG1 backbone, respectively. The anti-SSEA-4 p2-78 hAb wasproduced using FreeStyle 293 expression system and then purified throughthe protein G sepharose column. We examined the purity of antibody bySDS-PAGE analysis with coomassie blue staining (FIG. 13A). The resultshows the purity level of antibody exceed 95%. Subsequently, weperformed ELISA to investigate the binding activity of p2-78 hAb forGlobo-series glycans (FIG. 13B). We found that p2-78 hAb bound to SSEA-4and SSEA-3, but not to Globo H, which demonstrates the human IgG versionof p2-78 retains the activity of its parental scFv version to recognizethe binding epitope of SSEA-4.

We used glycan array containing 203 different glycans to further confirmthe specificity of p2-78 hAb. The results showed that p2-78 hAbrecognized SSEA4, Sialyl-SSEA4, SSEA4Gc, and Gb5 (SSEA3) (FIG. 14B).Interestingly, p2-78 hAb also recognized GloboH, similar to the resultsfrom ELISA assay (FIG. 12). The commercially available IgM antibody,MC631, was used as a positive control (FIG. 14A).

Development of Humanized MC48 mAbs

Non-humanized Murine mAbs may have certain limitations in clinicalsettings, including their short serum half-life, inability to triggerhuman effector functions and the production of human anti-murineantibodies (HAMA) response (LoBuglio et al., 1989). Therefore, mAbs canbe humanized by grafting their CDRs onto the VH and VL FRs of human Igmolecules (Roguska et al., 1994).

To develop humanized MC48, we sequenced V_(H) and V_(L) variable regionof MC48 from a hybridoma cell (Table 4). After alignment of V_(H) andV_(L) variable region of MC48 with the NCBI IgBLAST database, wemodified FRs of MC48 and generated 1^(st), 2^(nd), 3^(rd) and 4^(th)humanized MC48 sequences (Table 17, FIG. 17). We next constructed andgenerated the phage-displayed scFv formats according to these humanizedMC48 sequences. To determine the binding activity of the humanized MC48phage clones, we carried out solid-based ELISA coating SSEA-4-BSA (FIGS.15 and 18). We found that the humanized MC48 scFv phage could recognizeSSEA-4 in a dose-dependent manner. The data indicated that the 4^(th)humanized MC48 scFv phage maintained its binding affinity compared withthe murine mAb MC48.

Example 16: Complement-Dependent Cytotoxicity (CDC) Assay

The ability of exemplary humanized MC 48 to mediate CDC of SSEA-4expressing cells is examined. Homo sapiens breast or pancreaticcarcinoma cells were plated in each well of 96-well plates for growth ofovernight prior to the assay. The cells were then incubated withserially diluted concentrations of humanized MC 48 or human IgG1 isotypecontrol in RPMI in the presence of rabbit serum as a source ofcomplement (dilution of 1:5; Life Technologies). Cell death is assessedby the addition of the viability probe 7-AAD. Based on the results ofthe 7-AAD measurement, percentage-specific lysis is calculated using aFACScan flow cytometer. The antibodies show significant killing activityat 10 μg/mL compared to isotype control. As shown, humanized MC48-4successfully mediates CDC of SSEA-4 expressing cells.

Example 17: Materials and Methods

Construction of exemplary single chain fragments variable (scFv) ofMC41, 1^(st)-hMC41, 2^(nd)-hMC41 and 3^(rd)-hMC41 phage clones

The scFv form of MC41, 1^(st)-hMC41, 2^(nd)-hMC41 and 3^(rd)-hMC41sequences (V_(H)-GGGGSGGGGSGGGGS-V_(L)) were gene synthesized (Genomics)and cut by Sfi I and Not I (Fermentas). After gel extraction, thedigested products were cloned to pCANTAB-5E phagemid (GE Healthcare).hMC41 variant phagemids were transformed to TG1 E-coli and recovered in2×YT medium (BD Pharmingen) containing 100 μg/ml ampicillin and 2%glucose and rescued by M13KO7 helper phage (NEB) for 1 hour at 37° C.After centrifugation by 1,500×g for 10 min, these pellets wereresuspended in 2×YT medium containing 100 μg/ml ampicillin and 50 μg/mlkanamycin overnight to generate scFv-phages.

Demonstration of Efficacy: Binding Assay of MC41 and hMC41 scFv PhageClones or IgGs by ELISA

SSEA-4-BSA was coated on an ELISA plate at the concentration of 0.2μg/ml. After washing and blocking, the serial diluted phages or IgGswere incubated at RT for 1.5 hour. After washing, 1:1000 dilutedHRP-conjugated anti-M13 antibody (GE Healthcare), 1:2000 dilutedHRP-conjugated anti-human or -mouse IgG antibodies were added at RT for1 hour. Then, liquid substrate 3,3′,5,5′-tetramethylbenzidine (TMB)developed and was terminated with 3N HCl. Optical density was measuredat 450 nm.

Demonstration of Efficacy: Humanization of MC41

The two human genes, IGHJ4*08 and IGKV6-21*02, were the most similar toMC41 V_(H) and V_(L). As such, we chose FRs from these two genes forhumanization of MC41. CDR1 to CDR3 of V_(H) and V_(L) in all of thehumanized MC41 were conserved.

Demonstration of Efficacy: Construction and Expression of Anti-SSEA-4Humanized IgG

The V_(H) region of humanized MC41 was cloned with AgeI and NheI siteinto modified expression vector pcDNA5-FRT-Gamma1 containing a signalpeptide and the constant region of human immunoglobulin gamma 1 heavychain. The V_(L) region of humanized MC41 was cloned with AgeI and EcoRVsite into modified expression vector p-Kappa-HuGs containing a signalpeptide and constant region of human immunoglobulin kappa light chain.Both plasmids were transfected into FreeStyle293 cells (Invitrogen) andcontinuously incubated in serum-free medium at 37° C. for 1 week toproduce humanized antibody.

Demonstration of Efficacy: Purification of Anti-SSEA-4 Humanized IgG

The culture medium was collected, centrifuged and filtrated with 0.45 μmpore-size membrane. The supernatant then was subjected to protein Gcolumn chromatography (GE healthcare) for purification of anti-SSEA-4humanized IgG. After dialysis of eluents with PBS, the antibody wasexamined by SDS-PAGE analysis with coomassie blue staining as usual. Theconcentration of antibody was assessed by Bradford reagent (ThermoScientific) and spectrophotometer.

Demonstration of Efficacy: Binding Specificity of chMC41 and hMC41 byGlycan Array

Glycan array slides were blocked by 1% BSA for 45 min and then incubatedwith serially diluted chMC41 or hMC41 IgGs for another 45 mins at RT.After washing, donkey anti-human IgG Fcγ-F674 was used as secondantibody for 40 min at RT. Finally, the slides were washed, dried andsubsequently scanned with wavelength 674 nm.

Demonstration of Efficacy: Antibody-Dependent Cell Mediated Cytotoxicity(ADCC) Assay

HPAC (5×10³ cells) pancreatic cancer cell were seeded in a 96-well plateand cultured until ˜80% confluent. These cells were then incubated withantibodies chMC41, hMC41, MC813, NHIgG or NMIgG, together with PBMCs(effectors, E) at 37° C. for 16 hours. After treatment, the LDHexpression level was detected by CytoTox-ONE™ Homogeneous MembraneIntegrity Assay Kit (Promega). The reaction was read by fluorescencewith an excitation wavelength of 560 nm and emission wavelength of 590nm (Molecular Device, SpectraMax M5).

Demonstration of Efficacy: Complement-Dependent Cytotoxicity (CDC) Assay

HPAC (5×10³ cells) pancreatic cancer cell lines were cultured overnightto −80% confluent and reacted with mixture containing antibodies chMC41,hMC41, MC813, NHIgG or NMIgG and rabbit complement (20%) (Low-Tox-Mrabbit complement, Cedarlane) at 37° C. for 16 hours. Then, cellviability was measured by CytoTox-ONE™ Homogeneous Membrane IntegrityAssay Kit (Promega), following the same procedures as that of ADCCassay.

Demonstration of Efficacy: Development of Humanized MC41 mAbs

Murine mAbs have limited clinical use, including their short serumhalf-life, inability to trigger human effector functions and theproduction of human anti-murine antibodies (HAMA) response (LoBuglio etal., 1989). Therefore, mAbs have to humanize by grafting their CDRs ontothe V_(H) and V_(L) FRs of human Ig molecules (Roguska et al., 1994).

After alignment of V_(H) and V_(L) variable region of MC41 with the NCBIIgBLAST or IMGT database, we generated 1^(st), 2^(nd) and 3^(rd)humanized MC41 sequences. We next constructed and generated thephage-displayed scFv formats according to these humanized MC41sequences. To determine the binding activity of the humanized MC41 phageclones, we carried out solid-based ELISA coating SSEA-4-BSA (FIG. 1). Wefound 2^(nd) and 3^(rd) humanized MC41 scFv phages could recognizeSSEA-4 in a dose-dependent manner, whereas the 1^(st) MC41 scFv lost thebinding activity to SSEA-4 (FIG. 1). To evaluate the binding activity byintact humanized MC41 IgG, we constructed intact IgGs of 1^(st), 2^(nd),3^(rd) humanized MC41, and chimeric MC41 (chMC41). The ELISA resultsshowed that the humanized 2^(nd) and 3^(rd) MC41 could react to SSEA-4(FIG. 2A) but not to BSA (FIG. 2B) in a dose-dependent pattern, sameresults were observed for chMC41. The binding affinity of the 2^(nd) and3^(rd) humanized MC41 was maintained, compared to that of the murineMC41. We named humanized 2^(nd) IgG as hMC41. In order to determine thebinding specificity of chMC41 and hMC41, glycan array was performed. Thechimeric and humanized MC41 showed more specific binding than commercialSSEA4 antibody (MC813). They only recognized SSEA4 or glycolyl modifiedSSEA4 (FIG. 3).

Demonstration of Efficacy: ADCC and CDC of chMC41 and hMC41.

To demonstrate the effector function of chMC41 and hMC41, ADCC and CDCassays were performed. HPAC pancreatic cancer cell line was used toevaluate the ADCC and CDC activities of chMC41, hMC41, positive controlMC813 or negative controls NHIgG and NMIgG (FIGS. 4 and 5). The datashowed that the effector function of hMC41 was similar to chMC41.Interestingly, the humanized MC41 not only maintain its originalactivity, but it also showed stronger cancer cell killing activity thanMC813 through ADCC and CDC (FIG. 5).

REFERENCES

-   LoBuglio, A. F., Wheeler, R. H., Trang, J., Haynes, A., Rogers, K.,    Harvey, E. B., Sun, L., Ghrayeb, J., and Khazaeli, M. B. (1989).    Mouse/human chimeric monoclonal antibody in man: kinetics and immune    response. Proc Natl Acad Sci USA 86, 4220-4224.-   Roguska, M. A., Pedersen, J. T., Keddy, C. A., Henry, A. H.,    Searle, S. J., Lambert, J. M., Goldmacher, V. S., Blattler, W. A.,    Rees, A. R., and Guild, B. C. (1994). Humanization of murine    monoclonal antibodies through variable domain resurfacing. Proc Natl    Acad Sci USA 91, 969-973.

Example 18: Demonstration of Efficacy: Materials and Methods

Phage Display Biopanning Procedures

The phage-displayed human naive scFv library containing 2.5×10¹⁰ clones(Lu et al., 2011) was subtracted with non-specific binding inPEG-conjugated carboxyl Dynabeads (Invitrogen) at room temperature (RT)for 1 hour, and subsequently incubated with SSEA-4-PEG immobilizedDynabeads at 4° C. for 1 hour. After washing with PBS or PBS containing0.01% Tween 20 (PBST_(0.01)), the phages that bound toSSEA-4-PEG-Dynabeads were recovered by infection with E-coli TG1 cellsat 37° C. for 0.5 hour. Some of the infected cells were serially dilutedto determined titer, and the others were rescued by M13KO7 phage andamplified. After determination of rescued phages titer, the next roundof biopanning was performed. In the fourth and fifth round ofbiopanning, the phage clones were randomly selected to culture for ELISAscreening.

ELISA Screening of Selected Phage Clones

For detection of antigen recognition, microwell plates (Nunc) werecoated with 0.2 μg/ml of SSEA-4-BSA, Globo H-BSA, SSEA-3-BSA and BSA,respectively. The selected phage clones were diluted 1:2 in PBScontaining 3% BSA and added to each well. The plates were incubated atRT for 1 hour, washed with PBST_(0.1), and incubated with horseradishperoxidase (HRP)-conjugated mouse anti-M13 phage antibody (GEHealthcare). The plates were washed again, and OPD and H₂O₂ were added.After termination of reaction by 3 N HCl, the absorbance was measuredusing a 490 nm using microplate reader (Model 680, BioRad). We extractedphagemids from ELISA-positive phage clones to identity scFv codingregions by auto-sequencing.

Demonstration of Efficacy: Construction and Expression of Anti-SSEA-4Human IgG

The V_(H) region of selected scFv was cloned with AgeI and NheI siteinto modified expression vector pcDNA5-FRT-Gamma1 containing a signalpeptide and the constant region of human immunoglobulin gamma 1 heavychain. The V_(L) region of selected scFv was cloned with AgeI and EcoRVsite into modified expression vector p-Kappa-HuGs containing a signalpeptide and constant region of human immunoglobulin kappa light chain.Both plasmids were transfected into FreeStyle293 cells (Invitrogen) andcontinuously incubated in serum-free medium at 37° C. for 1 week toproduce human antibody.

Demonstration of Efficacy: Purification of Anti-SSEA-4 Human IgG

The culture medium was collected, centrifuged and filtrated with 0.45 μmpore-size membrane. The supernatant then was subjected to protein Gcolumn chromatography (GE healthcare) for purification of anti-SSEA-4human IgG. After dialysis of eluents with PBS, the antibody was examinedby SDS-PAGE analysis with coomassie blue staining as usual. Theconcentration of antibody was assessed by Bradford reagent (ThermoScientific) and spectrophotometer.

Demonstration of Efficacy: Humanization of MC48 and MC41

Two human genes, GenBank accession Q9UL73 and AY577298, were the mostsimilar to MC48 V_(H) and V_(L), respectively. We humanized threesequences of MC48, including the 1^(St) humanized MC48 (hMC48) V_(H)consisted of modified framework (FR) 1 to FR4 of Q9UL73 gene, the 1^(st)hMC48 V_(L) consisted of four FRs from the accession AY577298, the2^(nd) hMC48 FRs of V_(H) followed by 1YY8 from PDB, while the 2^(nd)hMC48 V_(L) same as 1^(st) sequence, and the 3^(rd) hMC48 V_(H) sequencemodified FR1, 2 and 4 of Q9UL73 gene and the 3^(rd) hMC48 V_(L) onlychanged FR2 and FR4 to human AY577298 gene. The other two human genes,IGHJ4*08 and IGKV6-21*02, were the most similar to MC41 V_(H) and V_(L).As such, we chose FRs from these two genes for humanization of MC41.CDR1 to CDR3 of V_(H) and V_(L) in all of the humanized MC48 and MC41were conserved.

Demonstration of Efficacy: Construction of Single Chain FragmentsVariable (scFv) of Humanized MC48 and MC41 Phage Clones

The scFv form of humanized MC48 (hMC48) and MC41 (hMC41) sequences(V_(H)-GGGGSGGGGSGGGGS-V_(L)) were gene synthesized (Genomics) and cutby Sfi I and Not I (Fermentas). After gel extraction, the digestedproducts were cloned to pCANTAB-5E phagemid (GE Healthcare). hMC48 andhMC41 variant phagemids were transformed to TG1 E-coli and recovered in2×YT medium (BD Pharmingen) containing 100 μg/ml ampicillin and 2%glucose and rescued by M13KO7 helper phage (NEB) for 1 hour at 37° C.After centrifugation by 1,500×g for 10 min, these pellets wereresuspended in 2×YT medium containing 100 μg/ml ampicillin and 50 μg/mlkanamycin overnight to generate scFv-phages.

Demonstration of Efficacy: Binding Assay of hMC48 and hMC41 scFv PhageClones or IgGs by ELISA

SSEA-4-BSA was coated on an ELISA plate at the concentration of 0.2μg/ml. After washing and blocking, the serial diluted phages or IgGswere incubated at RT for 1.5 hour. After washing, 1:1000 dilutedHRP-conjugated anti-M13 antibody (GE Healthcare), 1:2000 dilutedHRP-conjugated anti-human or -mouse IgG antibodies were added at RT for1 hour. Then, liquid substrate 3,3′,5,5′-tetramethylbenzidine (TMB)developed and was terminated with 3N HCl. Optical density was measuredat 450 nm.

Demonstration of Efficacy: Binding Specificity of p2-78 hAb, chMC41 andhMC41 by Glycan Array

Glycan array slides were blocked by 1% BSA for 45 min and then incubatedwith serially diluted p2-78 hAb, chMC41 or hMC41 IgGs for another 45mins at RT. After washing, donkey anti-human IgG Fcγ-F674 was secondantibody for 40 min at RT. Finally, the slides were washed, dried andsubsequently scanned with wavelength 674 nm.

Demonstration of Efficacy: Antibody-Dependent Cell Mediated Cytotoxicity(ADCC) Assay

HPAC, BxPC3 or PL45 (5×10³ cells) pancreatic cancer cell were seeded ina 96-well plate and cultured until ˜80% confluent. Then, these cellswere incubated with antibodies hMC48, hMC41 or NHIgG, together withPBMCs (effectors, E) at 37° C. for 16 hours. After treatment, the LDHexpression level was detected by CytoTox-ONE™ Homogeneous MembraneIntegrity Assay Kit (Promega). The reaction was read by fluorescencewith an excitation wavelength of 560 nm and emission wavelength of 590nm (Molecular Device, SpectraMax M5).

Demonstration of Efficacy: Complement-Dependent Cytotoxicity (CDC) Assay

HPAC, BxPC3 or PL45 (5×10³ cells) pancreatic cancer cell lines werecultured overnight to −80% confluent and reacted with mixture containingantibodies hMC48, hMC41 or NHIgG and rabbit complement (10% and 20%)(Low-Tox-M rabbit complement, Cedarlane) at 37° C. for 16 hours. Then,cell viability was measured by CytoTox-ONE™ Homogeneous MembraneIntegrity Assay Kit (Promega), following the same procedures as that ofADCC assay.

Demonstration of Efficacy:

Identification of Phage-Displayed scFv that Binds to SSEA-4

To identify the antibodies that bind to SSEA-4, we used phage-displayedhuman naive scFv library containing 2×10¹⁰ members, which wasestablished as described in our previous report (Lu et al., 2011). Thislibrary was first removed by Dynabeads-binding phages, and thenSSEA-4-binding phages were selected by SSEA-4-PEG-conjugated Dynabeads.We used two buffer systems, PBS and PBS containing 0.01% Tween20(PBST_(0.01)), during biopanning. After five rounds of affinityselection, the phage recovery of the fifth round increased by about55-fold and 80-fold, compared to that of the first round in PBS andPBST_(0.01) system, respectively (FIG. 1). The phage clones wererandomly selected and tested for SSEA-4 binding by ELISA (FIG. 2). Wefound seven clones that specifically bound to SSEA-4-BSA, but not to BSAcontrol protein. By sequencing all 8 individual clones, we identifiedtwo unique anti-SSEA-4 phage clones (p1-52 and p2-′78) which containeddistinct human V_(H) and V_(L) coding regions (Table 1).

To examine the specificity and binding affinity of the two phage clones,we performed a comparative ELISA using the same phage titer toGlobo-series glycans including SSEA-4-BSA, Globo H-BSA and SSEA-3-BSA(FIG. 3). The p2-78 phage clone showed the strong binding to SSEA-4-BSAand SSEA-3-BSA, and more slight binding to Globo H-BSA. However, wefound that the binding activity of p1-52 phage clone to SSEA-4-BSA wasvery weak. Thus we focused on p2-78 clone for further study.

To establish the fully human antibody (hAb) against SSEA-4, wemolecularly engineered the V_(H) and V_(L) coding sequences of p2-78scFv into human IgG₁ backbone, respectively. The anti-SSEA-4 p2-78 hAbwas produced using FreeStyle 293 expression system and then purifiedthrough the protein G sepharose column. We examined the purity ofantibody by SDS-PAGE analysis with coomassie blue staining (FIG. 4A).The result shows the purity level of antibody exceed 95%. Subsequently,we performed ELISA to investigate the binding activity of p2-78 hAb forGlobo-series glycans (FIG. 4B). We found that p2-78 hAb bound to SSEA-4and SSEA-3, but not to Globo H, demonstrating that the human IgG versionof p2-78 retains the activity of its parental scFv version to recognizethe binding epitope of SSEA-4.

We used glycan array containing 203 different glycans to further confirmthe specificity of p2-78 hAb. The results showed that p2-78 hAbrecognized SSEA4, Sialyl-SSEA4, SSEA4Gc, and Gb5 (SSEA3) (FIG. 5B).Interestingly, p2-78 hAb also slightly recognized Globo H, similar tothe results from ELISA assay (FIG. 3). The commercially available IgMantibody, MC631, was used as a positive control (FIG. 5A).

Demonstration of Efficacy: Development of Humanized MC48 and MC41 mAbs

Murine mAbs have limited clinical use, including their short serumhalf-life, inability to trigger human effector functions and theproduction of human anti-murine antibodies (HAMA) response (LoBuglio etal., 1989). Therefore, mAbs have to humanize by grafting their CDRs ontothe V_(H) and V_(L) FRs of human Ig molecules (Roguska et al., 1994).

After alignment of V_(H) and V_(L) variable region of MC48 and MC41 withthe NCBI IgBLAST or IMGT database, we generated 1^(st), 2^(nd), 3^(rd)and 4^(th) humanized, MC48 sequences and 1^(st), 2^(nd) and 3^(rd)humanized MC41 sequences. We next constructed and generated thephage-displayed scFv formats according to these humanized MC48 and MC41sequences. To determine the binding activity of the humanized MC48 andMC41 phage clones, we carried out solid-based ELISA coating SSEA-4-BSA(FIGS. 6, 7 and 8). We found that the 3^(rd) and 4^(th) humanized MC48,and 2^(nd) and 3^(rd) humanized MC41 scFv phages could recognize SSEA-4in a dose-dependent manner, whereas the 1^(st) and 2^(nd) humanized MC48and 1^(st) MC41 scFv lost the binding activity to SSEA-4 (FIGS. 6, 7 and8). The data showed that the binding affinities of the 4^(th) humanizedMC48, and 3^(rd) humanized MC41 scFv phage clones were maintained,compared to that of the murine mAbs MC48 or MC41. To evaluate thebinding activity by intact humanized MC41 IgG, we constructed intactIgGs of 1^(st), 2^(nd), 3^(rd) humanized MC41 and chimeric MC41(chMC41). The ELISA results showed that the humanized 2^(nd) and 3^(rd)MC41 could react to SSEA-4 (FIG. 9A) but not to BSA (FIG. 9B) in adose-dependent pattern, same results were observed for chMC41. We namedhumanized 2^(nd) IgG as hMC41. In order to determine the bindingspecificity of chMC41 and hMC41, glycan array was performed. Thechimeric and humanized MC41 showed more specific binding than commercialSSEA4 antibody (MC813). They only recognized SSEA4 or glycolyl modifiedSSEA4 (FIG. 10).

Demonstration of Efficacy: ADCC and CDC Test of hMC48, chMC41 and hMC41

To investigate the effector function of hMC48, chMC41 and hMC41, ADCCand CDC assays were performed. HPAC, BxPC3 and PL45 pancreatic cancercell lines were used to evaluate the ADCC and CDC activities at theconcentration of 10 μg/ml for hMC48 or NHIgG (FIG. 11). Further, HPACcells were treated with chMC41, hMC41, positive control MC813 ornegative control NHIgG (FIGS. 12 and 13). The data showed that theeffector function of hMC41 and chMC41 was superior to that of hMC48.Interestingly, the humanized MC41 not only maintain its originalactivity, but it also showed stronger cancer cell killing activity thanMC813 through ADCC and CDC (FIG. 13).

Example 19: Binding of MC41 vs MC 48

The binding abilities of hMC41 and hMC48 to SSEA-4 were examined byELISA. The result showed that the binding of hMC41 to SSEA-4 was muchbetter than hMC48. The humanized MC41 has a higher binding maximum and asmaller Kd (0.2 μg/ml and 4.6 μg/ml for hMC41 and hMC48, respectively)value as compared to hMC48.

REFERENCES

-   LoBuglio, A. F., Wheeler, R. H., Trang, J., Haynes, A., Rogers, K.,    Harvey, E. B., Sun, L., Ghrayeb, J., and Khazaeli, M. B. (1989).    Mouse/human chimeric monoclonal antibody in man: kinetics and immune    response. Proc Natl Acad Sci USA 86, 4220-4224.-   Lu, R.-M., Chang, Y.-L., Chen, M.-S., and Wu, H.-C. (2011). Single    chain anti-c-Met antibody conjugated nanoparticles for in vivo    tumor-targeted imaging and drug delivery. Biomaterials 32,    3265-3274.-   Roguska, M. A., Pedersen, J. T., Keddy, C. A., Henry, A. H.,    Searle, S. J., Lambert, J. M., Goldmacher, V. S., Blattler, W. A.,    Rees, A. R., and Guild, B. C. (1994). Humanization of murine    monoclonal antibodies through variable domain resurfacing. Proc Natl    Acad Sci USA 91, 969-973.

What is claimed is:
 1. An isolated monoclonal antibody orantigen-binding fragment thereof that binds toNeu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, wherein the antibody orantigen-binding fragment thereof comprises a glycan attached to Asn-297of the Fc region, wherein the glycan has the formula: Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂, as in the structure shown in FIG. 1, andwherein the antibody or antigen-binding fragment thereof comprises anH-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2, and an L-CDR3,wherein: (i) the H-CDR1 comprises the sequence of SEQ ID NO:152(GFSLTSYG); (ii) the H-CDR2 comprises the sequence of SEQ ID NO:153(IWGEGST); (iii) the H-CDR3 comprises the sequence of SEQ ID NO:154(AMTGTAY); (iv) the L-CDR1 comprises the sequence of SEQ ID NO:149(SSVSY); (v) the L-CDR2 comprises the sequence of SEQ ID NO:150 (DTS);and (vi) the L-CDR3 comprises the sequence of SEQ ID NO:151(HQWSSSPHT).2. The isolated monoclonal antibody or antigen-binding fragment thereofof claim 1, wherein the antibody is an IgG1.
 3. The isolated monoclonalantibody or antigen-binding fragment thereof of claim 2 wherein theantibody comprises a VH having SEQ ID NO:147 and a VL having SEQ IDNo:148.
 4. The isolated monoclonal antibody or antigen-binding fragmentthereof of claim 1 wherein the antibody is a humanized antibody.
 5. Apharmaceutical composition comprising the isolated monoclonal antibodyor antigen-binding fragment thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 6. The isolated monoclonal antibody orantigen-binding fragment thereof of claim 2, wherein the antibodycomprises a VH having SEQ ID NO:137 and a VL having SEQ ID NO:138. 7.The isolated monoclonal antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody or antigen-binding fragment thereoffurther comprises an H-FR1, an H-FR2, an H-FR3, an HFR4, an L-FR1, anL-FR2, an L-FR3, and an L-FR4, wherein: (i) the H-FR1 comprises thesequence of SEQ ID NO: 159 (QVQLKESGPGLVAPSQSLSITCTVS); (ii) the H-FR2comprises the sequence of SEQ ID NO:160 (VSWIRQPPGKGLEWIGV); (iii) theH-FR3 comprises the sequence of SEQ ID NO:161(NYHSVLISRLTISKDNSKSQVFLKLNSLQTDDTATYYC); (iv) the H-FR4 comprises thesequence of SEQ ID NO:162 (WGQGTLVTVSS); (v) the L-FR1 comprises thesequence of SEQ ID NO:155 (QIVLTQSPAIMSASPGEKVTMTCSAS); (vi) the L-FR2comprises the sequence of SEQ ID NO 156 (MHWYQQKSGTSPKRWIY); (vii) theL-FR3 comprises the sequence of SEQ ID NO:157(KLSSGVPGRFSGSGSGTSYSLTISRLEAEDAATYYC); and (viii) the L-FR4 comprisesthe sequence of SEQ ID NO:158 (FGGGTKVEIKR).
 8. An isolated monoclonalantibody or antigen-binding fragment thereof that binds toNeu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, wherein the antibody orantigen-binding fragment thereof comprises a glycan attached to Asn-297of the Fc region, wherein the glycan has the formula:Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂, as in the structure shown in FIG. 1,and wherein the antibody or antigen-binding fragment thereof comprisesan H-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2, and an L-CDR3,wherein: (i) the H-CDR1 comprises the sequence of SEQ ID NO:207; (ii)the H-CDR2 comprises the sequence of SEQ ID NO:208; (iii) the H-CDR3comprises the sequence of SEQ ID NO:209; (iv) the L-CDR1 comprises thesequence of SEQ ID NO:204; (v) the L-CDR2 comprises the sequence of SEQID NO:205; and (vi) the L-CDR3 comprises the sequence of SEQ ID NO:206.9. The isolated monoclonal antibody or antigen-binding fragment thereofof claim 8, wherein the antibody is a humanized antibody.
 10. Theisolated monoclonal antibody of claim 8, wherein the monoclonal antibodyor antigen-binding fragment thereof comprises a VH having SEQ ID NO:202and a VL having SEQ ID No:203.
 11. A pharmaceutical compositioncomprising the isolated monoclonal antibody or antigen-binding fragmentthereof of claim 8 and a pharmaceutically acceptable carrier.
 12. Anisolated monoclonal antibody or antigen-binding fragment thereof thatbinds to Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1, wherein theantibody or antigen-binding fragment thereof comprises a glycan attachedto Asn-297 of the Fc region, wherein the glycan has the formula:Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂, as in the structure shown in FIG. 1,and wherein the antibody or antigen-binding fragment thereof comprisesan H-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2, and an L-CDR3,wherein: (i) the H-CDR1 comprises the sequence of SEQ ID NO:217; (ii)the H-CDR2 comprises the sequence of SEQ ID NO:218; (iii) the H-CDR3comprises the sequence of SEQ ID NO:219; (iv) the L-CDR1 comprises thesequence of SEQ ID NO:214; (v) the L-CDR2 comprises the sequence of SEQID NO:215; and (vi) the L-CDR3 comprises the sequence of SEQ ID NO:216.13. The isolated monoclonal antibody or antigen-binding fragment thereofof claim 12, wherein the antibody is a humanized antibody.
 14. Theisolated monoclonal antibody or antigen-binding fragment thereof ofclaim 12, wherein the antibody comprises a VH having SEQ ID NO:212 and aVL having SEQ ID NO:213.
 15. A pharmaceutical composition comprising theisolated monoclonal antibody or antigen-binding fragment thereof ofclaim 12 and a pharmaceutically acceptable carrier.
 16. An isolatedmonoclonal antibody or antigen-binding fragment thereof that binds toNeu5Acα2→3Galβ1→3GalNAcβ1→3Galαl→4Galβ1→4Glcβ1, wherein the antibody orthe antigen-binding fragment thereof comprises a glycan attached toAsn-297 of the Fc region, wherein the glycan has the formula:Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂, as in the structure shown in FIG. 1,and wherein the antibody or antigen-binding fragment thereof comprisesan H-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2, and an L-CDR3,wherein: (i) the H-CDR1 comprises the sequence of SEQ ID NO:227; (ii)the H-CDR2 comprises the sequence of SEQ ID NO:228; (iii) the H-CDR3comprises the sequence of SEQ ID NO:229; (iv) the L-CDR1 comprises thesequence of SEQ ID NO:224; (v) the L-CDR2 comprises the sequence of SEQID NO:225; and (vi) the L-CDR3 comprises the sequence of SEQ ID NO:226.17. The isolated monoclonal antibody or antigen-binding fragment thereofof claim 16, wherein the antibody is a humanized antibody.
 18. Theisolated monoclonal antibody or antigen-binding fragment thereof ofclaim 16, wherein the antibody comprises a VH having SEQ ID NO:222 and aVL having SEQ ID NO:223.
 19. A pharmaceutical composition comprising theisolated monoclonal antibody or antigen-binding fragment thereof ofclaim 16 and a pharmaceutically acceptable carrier.